Adenovirus-mediated transfer of siRNA against MMP-2 mRNA results in impaired invasion and tumor-induced angiogenesis, induces apoptosis in vitro and inhibits tumor growth in vivo in glioblastoma

Invasive tumors, including gliomas, utilize proteinases to degrade extracellular matrix components and diffuse into the adjacent tissues or migrate toward distant ones. In addition, proteinase activity is required for the formation of new blood vessels within the tumor. Levels of the proteinase matrix metalloproteinase-2 (MMP-2) are highly increased in gliomas. In this study, we examined the effect of the downregulation of MMP-2 via adenovirus-mediated siRNA in gliomas. Here, we show that siRNA delivery significantly decreased levels of MMP-2 in the glioblastoma cell lines U-87 and U-251. U-87 and U-251 cells showed impaired invasion through matrigel as well as decreased migration from tumor spheroids transfected with adenoviral vector expressing siRNA against MMP-2. Additionally, tumor-induced angiogenesis was decreased in in vitro experiments in cultured human microvascular endothelial cells (HMECs) in serum-free conditioned medium of glioblastoma cells transfected with these constructs and co-cultures of glioma cells with HMECs. We also observed decreased angiogenesis in the in vivo dorsal skin-fold chamber model. Moreover, MMP-2 inhibition induced apoptotic cell death in vitro, and suppressed tumor growth of preestablished U-251 intracranial xenografts in nude mice. Thus, specific targeting of MMP-2 may provide a novel, efficient approach for the treatment of gliomas and improve the poor outcomes of patients with these brain tumors.     

Risk of cancer revealed by follow-up of families with hereditary non-polyposis colorectal cancer: Reply to Dr. Eluf-Neto

Congenitally athymic nude (nu/nu) mice have been used extensively as hosts to study the metastatic properties of xenografted human tumors. It has not been fully explored to what extent the metastatic behavior of human tumor xenografts reflects the intrinsic properties of the tumor cells and to what extent it is influenced artificially by the host. The purpose of the work reported here was to evaluate the potential usefullness of athymic mice for qualitative and quantitative assessment of the intrinsic metastatic properties of human tumor cells. Four human melanoma cell lines (A-07, D-12, R-18, U-25) were included in the study. Quantitative intradermal and intracranial transplantation assays were used to determine the tumorigenicity and immunogenicity of the cell lines. The metastatic behavior of the cell lines was studied by inoculating cells intra-arterially or intravenously (artificial metastases) or intra-dermally (spontaneous metastases). The influence of the host on the incidence of metastases was assessed by inoculating cells intravenously in immunologically modified athymic mice. Both the intradermal and intracranial tumorigenicity differed substantially between the cell lines. The host immune reactivity against heterotransplantation was significantly lower for D-12 than for A-07, R-18 and U-25 cells. The incidence of metastases was influenced significantly by the host immune reactivity. The cell lines showed organ-specific metastatic patterns in athymic mice. The organs of preference were lungs for A-07 and D-12 cells, lymph nodes for R-18 cells, and brain for U-25 cells. The organ-specific metastatic pattern in athymic mice reflected the pattern of distant metastases in the donor patients. In addition, all cell lines gave rise to metastases in abdominal organ sites, but without organ specificity. The incidence of abdominal metastases was influenced significantly by the tumorigenicity of the cell lines. In conclusion, athymic mice appear to be excellent hosts for assessment of the organ specificity in the metastatic behavior of human tumors. The metastasis frequency of human tumors in athymic mice, however, might be a misleading quantitative measure of the intrinsic metastatic propensity of the tumor cells, owing to the cell-line-dependent host immune reactivity. © 1995 Wiley-Liss, Inc. © 1995 Wiley-Liss, Inc.     

Comparative effect of oncolytic adenoviruses with E1A-55 kDa or E1B-55 kDa deletions in malignant gliomas

Replication-competent oncolytic adenoviruses hold considerable promise for treating malignant gliomas. The toxicity of the clinically tested E1B-55 kDa mutant virus is negligible; however, its full clinical potential is still being evaluated. The purpose of the present study is to compare the antiglioma activity in vitro and in vivo between Delta-24, an E1A mutant adenovirus, and RA55, an E1B-55 kDa mutant adenovirus. We selected human glioma cell lines that were tumorigenic in nude mice and express wild-type p53 (U-87 MG, D54 MG) or mutant p53 (U-251 MG, U-373 MG) protein. Our studies demonstrated that Delta-24 induced a more potent antiglioma effect in vitro than RA55. Moreover, Delta-24 replicated markedly more efficiently than RA55 in both wild-type and mutant p53 scenarios. Importantly, direct intratumoral injection of Delta-24, but not RA55, significantly suppresses tumor growth in intracranial (U-87 MG, U-251 MG) or subcutaneous (D54 MG) animal models. Staining for hexon protein detected replicating adenoviruses in xenografts infected with Delta-24, but not with RA55. Collectively, these data indicate that E1A mutant adenoviruses targeting the Rb pathway are more powerful putative agents for antiglioma therapy than E1B mutant adenoviruses, and suggest that E1A mutant adenoviruses should be tested in the clinical setting for patients with malignant gliomas.     

Study on human lymphoblastoid interferon-alpha (MOR-22): Part III. Antitumor effect on nude mouse-transplanted human tumors

The effects of human lymphoblastoid interferon-alpha (MOR-22) on the growth of xenografted human tumors in nude mice were examined. IFN-alpha was administered subcutaneously to mice with renal tumor (ACHN), intratumorally for glioblastoma (U-373MG) or intravenously with uterine cervical tumor (HeLa S3). The dosages of IFN-alpha were 2 X 10(4)-5 X 10(5) IU/mouse for ACHN tumor, 1 X 10(5)-5 X 10(5) IU/mouse for U-373MG tumor and 3 X 10(4)-1 X 10(5) IU/mouse for HeLa S3 tumor. IFN-alpha inhibited the growth of these tumors in a dose-dependent manner.     

Laminin adhesion-selected primary human colon-cancer cells are more tumorigenic than the parenteral and nonadherent cells

Laminin has been shown to promote the malignant phenotype and the level of the 32/67 Kd laminin receptor has been found to correlate with Dukes' staging of colon cancer. A biopsy of a Dukes' stage B2 human colon carcinoma formed a tumor in a nude mouse after coinjection with Matrigel. The parental tumor and the murine tumor appeared identical at the histological level. A cell line LCC-C1 was established from the murine tumor. The cell line appeared moderately differentiated although it did not produce mucin in vitro; however, the xenograft in vivo did produce low levels of mucin. Laminin adherent and non-adherent cell lines were selected. The parental and the laminin-selected cell subclones adhered equally well to plastic and to fibronectin and showed similar growth rates on plastic. When injected subcutaneously into nude mice, the laminin-adherent cells formed relatively undifferentiated tumors that were twice as large as the parental cell tumors whereas the laminin non adherent cells formed very small, but highly differentiated tumors. These data demonstrate that subpopulations of tumor cells which differ in their tumorigenic properties can be selected based on their adhesion to laminin and thus provide models for studying the mechanisms of tumor growth.     

Transplantation of human prostatic carcinoma into nude mice in Matrigel

Previous successful transplantation of human primary prostatic carcinomas into nude mice has been described as "close to zero." When injected in Matrigel instead of culture medium, 25,000-fold fewer cells of the PC-3 human prostatic carcinoma cell line were required for the growth of tumors in nude mice during a 3-month period of observation; similar enhancement was observed with two other human prostatic carcinoma cell lines. Six of ten primary human prostatic carcinomas were transplanted successfully into nude mice when Matrigel was used as the vehicle.     

Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate

The activity of the serine protease plasminogen activator (PA), which correlates with tumorigenicity and metastatic capacity, was examined using the 125I-labeled fibrin plate assay in cell extracts from four human glioma lines as a function of growth in vitro. Cell-associated inhibitory activity to plasmin and urokinase-type PA was also measured concurrently. The relative PA activities differed markedly among the lines, whereas inhibitory activities did not. Two lines, SNB-19 and SNB-75, exhibited maximal PA activities (1-6 m Plough units/micrograms protein) as cultures approached confluence, whereas two other lines, SNB-56 and SNB-78, expressed low PA activity at all times (less than 0.2 m Plough units/micrograms protein). The PA of SNB-19 cell extracts was predominantly urokinase-type PA. In addition to having the highest PA levels, SNB-19 and SNB-75 were the most clonogenic in soft agar and tumorigenic in nude mice. In contrast, SNB-56 and SNB-78 were poorly clonogenic in soft agar and were not tumorigenic in nude mice. Measured directly, inhibitory activities to plasmin, urokinase-type PA, and tissue-type PA were detected in SNB-19 (high PA) and SNB-56 (low PA) cell extracts. However, there were no qualitative or quantitative differences in inhibitor effects between SNB-19 and SNB-56 suggesting that the differences in PA activity between these lines resulted from changes in PA activity and were not due to differential plasminogen activator inhibitor effects. The ability of the differentiating agent sodium butyrate (NaB) to modulate total PA activity was also examined. Peak SNB-19 cell PA activity was decreased in a concentration (Ki, 0.75 mM) and time-dependent manner by the addition of nontoxic amounts of NaB. The dose-dependent decrease in PA activity induced by NaB was most likely due to an effect on PA itself, since the action of inhibitor on urokinase was unchanged in response to NaB. These results suggest that net cellular PA activity in glioma cells is a balance between relative PA activity and inhibitor(s) effects and that this balance can be modulated by sodium butyrate.     

The inhibition of neoplastic cell proliferation with human natural tumor necrosis factor

Purified human natural tumor necrosis factor (n-TNF) was prepared by stimulating human leukemic B cell line (BALL-1) with Sendai virus. The colony formations of all of 18 human cancer-derived abnormal cell lines were suppressed by 10(1)-10(6) U/ml of n-TNF, while n-TNF was nontoxic to all human normal fibroblast cells. This in vitro inhibition of cell growth was reversible. In breast adenocarcinoma MCF7 cells treated with n-TNF a specific decrease of DNA synthesis was observed, and DNA histograms showed a block at G1 in the cell cycle. In vivo studies revealed that n-TNF suppressed the tumor growth of murine Meth A sarcoma, human renal adenocarcinoma (ACHN), malignant melanoma (SK-MEL-28) and glioblastoma (U-373 MG). Isobologram analysis showed that n-TNF synergistically inhibited cell growth in combination with human natural interferon (IFN)-a. In vivo synergism of n-TNF and IFN-a was also found in the U-373 MG tumor model implanted into nude mice.     

The invasive features of glial and non-central nervous system tumor cells are different on organotypic brain slices from newborn rats

On radiograms, glial tumors are usually seen to invade in a finger-like fashion, while non-central nervous system (CNS) tumors expand in a mass-like fashion. We prepared organotypic brain slice cultures from newborn rats to investigate the invasive behavior of human brain tumors using glial tumor cell lines (U-87MG, U-373MG, U-251MG, and SF-126) and of non-CNS tumors using cell lines; HT-1080 (human malignant fibrosarcoma), RFRF (human lung carcinoma), MIA-PICA (human pancreatic carcinoma), and Colo38 (human malignant melanoma). We selected an area that was centered at a junction between deep cortical layers and the striatum, punched a hole measuring 0.5-0.7 mm in diameter in this area, implanted different rhodamine-labeled tumor cells at that site, and observed their invasive behavior under an inverted fluorescent microscope. Over the course of several days, all glioma cells invaded in a finger-like fashion; U-373 MG cells invaded farthest. Non-CNS tumors expanded in mass-like fashion into adjacent areas. Using the slice cultures, we also investigated the regulatory effect on tumor invasion of forced expression of glial fibrillary acidic protein (GFAP) after gene transfection. The forced expression of GFAP rendered U-87MG and HT-1080 cells less invasive. Our results show that organotypic brain slice cultures are an excellent medium for studying the invasive features of glial and non-CNS tumors.     

Human breast cancer cell line xenografts as models of breast cancer — The immunobiologies of recipient mice and the characteristics of several tumorigenic cell lines

Summary The ability to maintain and study human tissues in anin vivo environment has proved to be a valuable tool in breast cancer research for several decades. The most widely studied tissues have been xenografts of established human breast cancer cell lines into athymic nude mice. Human breast tumor xenografts provide the opportunity to study various important interactions between the tumor and host tissues, including endocrinologic, immunologic, and tumor-stroma interactions. The nude mouse is not the only immune-deficient recipient system in which to study xenografts. Additional single and combined mutant strains have been used successfully, including mice homozygous for the severe combined immune deficiency mutation (scid), both the beige (bg) and nude (nu) mutations in combination (bg/nu), and mice bearing the combinedbg/nu/xid mutations. The differing immunobiologies are discussed, with particular reference to the immunobiology of breast cancer, as are the characteristics of several of the more frequently utilized breast cancer xenografts and cell lines. The ability of several endocrine treatments to modulate effectors of cell mediated immunity,e.g., estrogens and antiestrogens, and the effect of site of inoculation on tumor take and metastasis, also are described.     

Increased tumorigenicity of human keratinocytes harboring human papillomavirus type 16 is associated with resistance to endogenous tumor necrosis factor-α-mediated growth limitation

The aim of this study was to evaluate the relationship between tumorigenicity of cell sublines derived from weakly tumorigenic SKv-e and SKv-I keratinocytes harboring human papillomavirus type 16 (HPV 16) and their susceptibility to autocrine growth limitation mediated by tumor necrosis factor-α (TNF-α). These sublines displayed different in vitro proliferative potential which correlated with tumorigenicity in nu/nu mice. Recombinant TNF-α inhibited in vitro growth of weakly tumorigenic parental SKv cell lines while it did not affect proliferation of their respective highly tumorigenic sublines. Resistance to TNF-α correlated with both increased in vitro proliferation and tumorigenicity. Anti-TNF-α antibodies (Ab) significantly increased in vitro proliferation of weakly tumorigenic parental SKv cells up to the levels of their highly tumorigenic sublines. Growth of highly tumorigenic SKv cells was not affected. On the other hand, proliferation of SKv cells was affected neither by transforming growth factor-β (TGF-β) nor by anti-TGF-β Ab. All SKv cell sublines tested spontaneously released TNF-α, as evaluated by a specific radioimmunoassay; however, the levels of the endogenous cytokine were not related to their proliferative potential and tumorigenicity. An increased resistance to the anti-proliferative effect of TNF-α may be associated with decreased expression of TNF-α receptors (TNF-αR) inasmuch as evaluation of ¹²⁵ I-TNF-α binding and Northern-blot analysis of TNF-αR-specific mRNA showed that highly tumorigenic SKv cell sublines expressed significantly lower numbers of TNF-αR than their respective parental cells. These results show that an increased tumorigenicity of HPV 16-harboring SKv keratinocytes may be, at least partially, due to escape from autocrine TNF-α-mediated growth limitation.     

Camptothecin derivatives induce regression of human ovarian carcinomas grown in nude mice and distinguish between non-tumorigenic and tumorigenic cells in vitro

We have recently shown that the plant alkaloid 20(S)-camptothecin and its derivatives 9-nitro-20(S)-camptothecin(9NC) and 9-amino-2O(S)-camptothecin(9AC) inhibit the growth of a variety of human tumors xenografted in nude mice. In this report, we demonstrate that 9NC and 9AC effectively inhibit growth, and subsequently induce regression, of human ovarian tumors grown in nude mice. Tumor regression is accompanied by degenerative changes in the tumor cells as assessed by microscopic observations of histological sections prepared from the tumors. Parallel experiments in vitro show that 9NC inhibits in a similar manner the growth of human ovarian carcinoma cells, regardless of their ability to induce tumors when xenografted in nude mice, and induces similar morphological changes in both non-tumorigenic and tumorigenic cells, as assessed by microscopic observation. Flow cytometry studies show that 9NC-induced growth inhibition of the non-tumorigenic cells is associated with accumulation of these cells in G₂. In contrast, 9NC-induced growth inhibition of the tumorigenic cells is associated with the generation of cells containing a reduced DNA content, that is, cells programmed to die. In conclusion, camptothecins appear to be cytostatic for non-tumorigenic, but cytotoxic for tumorigenic cells, an important finding from viewpoints of cell biology, pharmacology and cancer chemotherapy.     

Stimulation of human prostatic carcinoma tumor growth in athymic mice and control of migration in culture by extracellular matrix.

The tumorigenicity, migration, growth and invasiveness of certain tumor cells is stimulated by basement membranes. Here we have examined the effect of Matrigel, an extract of basement membrane proteins, on the behavior of several prostate cancer cell lines, testing their growth and invasiveness in vitro and in vivo. Cells of the Tsu-prI line were more invasive than PC-3, Du-145, or LNCaP cells. Peptide inhibitors implicated laminin in the migration and invasion of these cells. When these cells were suspended in Matrigel and injected into nude mice, their growth was greatly enhanced, since large tumors formed in athymic nude mice whereas virtually no tumors were observed in the absence of Matrigel. The growth of a slowly growing line, LNCaP, was increased by exogenous basic fibroblast growth factor when injected with Matrigel. A laminin cell adhesion peptide, YIGSR, was a potent inhibitor of Matrigel-stimulated tumor growth implicating cell-laminin interactions in this process. These results suggest that tumor growth of prostate adenocarcinoma cells may be dependent both on cellular growth factors and on cell-matrix interactions mediated by laminin which facilitate the development of transplanted tumors in nude mice.     

Stimulation of angiogenesis as an explanation of matrigel-enhanced tumorigenicity

Matrigel, a reconstituted extract of basement membrane, enhances the growth of different human cancer cell lines when transplanted into nude mice. Here that stimulation was confirmed in the BALB/c murine mammary-tumor cell line M3MC, as well as in human colon (SW948) and mammary (MDA-MB-468) carcinoma cell lines transplanted in nude and SCID mice, respectively. Subcutaneous and intra-mammary fat-pad inoculations of Matrigel alone generated an angiogenic response which was macroscopically evident by day 9. Histological analysis of the local host reaction occurring at the site of injection revealed an early peripheral fibroblast response, followed by mononuclear cell infiltration, solid and hollow fibroblast cords projections from the edge to the center of the Matrigel plug, and finally capillary ingrowths. Conditioned media obtained from the gels generated in vivo, acted as very strong chemoattractants for mouse lung capillary endothelial cells, stimulating their motility between 38 and 82 times with respect to the control. Our results suggest an important role of host cells recruited by Matrigel, which could favor angiogenesis of the area and thus facilitate the growth of tumor cells co-inoculated with the basement membrane extract.     

Tumorigenicity of human breast cancer is associated with loss of the Ca2+-activated chloride channel CLCA2.

The human Ca2+-activated chloride channel-2 (CLCA2) is expressed in normal breast epithelium but not in breast tumors of different stages of progression. Northern analysis of nontransformed and transformed breast epithelial cell lines revealed CLCA2 expression in the nontransformed cell line MCF10A and the nontumorigenic cell line MDA-MB-453, whereas all tumorigenic cell lines were negative (MDA-MB-231, MDA-MB-435, MDA-MB-468, and MCF7). When stably reintroduced into CLCA2-negative MDA-MB-231 and MDA-MB-435 cells, CLCA2 expression reduced Matrigel invasion in vitro and inducibility of s.c. and metastatic tumors of MDA-MB-231 cells in nude mice. Our results suggest that CLCA2 may act as a tumor suppressor in breast cancer.     

VEGF,bFGF and EGF in the angiogenesis of human melanoma xenografts

Vascular endothelial growth factor (VEGF) is a major inducer of angiogenesis in tumors. Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have both been shown to interact with VEGF. The involvement of VEGF, bFGF and EGF in melanoma angiogenesis was investigated here. Four human melanoma cell lines (A-07, D-12, R-18, U-25) were included in the study. Angiogenesis was quantified by scoring of tumor-oriented capillaries following intradermal cell inoculation in BALB/c nu/nu mice. VEGF, bFGF and EGF expression and secretion were investigated by Western blotting and enzyme-linked immunosorbent assay, respectively. Immunohistochemistry of xenografts grown intradermally was used to reveal VEGF and bFGF localization in vivo. The rate of angiogenesis differed substantially among the melanoma lines; the sequence from a high to low rate of angiogenesis was: A-07, D-12, R-18, U-25. A-07, which induced the highest rate of angiogenesis, showed a higher rate of VEGF secretion, stronger VEGF staining by immunohistochemistry and higher bFGF expression than the other lines. U-25, which induced the lowest rate of angiogenesis, showed a higher rate of VEGF secretion than D-12 and R-18. A-07 was the only line that showed detectable bFGF secretion, and R-18 was the only line that showed detectable EGF secretion. VEGF is probably important in the angiogenesis of melanomas. However, heterogeneity in rate of angiogenesis among melanomas cannot be attributed to heterogeneity in rate of secretion of VEGF, bFGF and/or EGF only. Int. J. Cancer 76:836–841, 1998.© 1998 Wiley-Liss, Inc.     

Characterization of tumorigenic sub-lines from a poorly tumorigenic human colon-adenocarcinoma cell line

The interaction of tumor cells with extracellular-matrix components is suspected to play an important role in tumorigenesis induction. The tumorigenicity of a poorly tumorigenic human colon-adenocarcinoma cell line (BCS-TC2) was induced by co-injection with Matrigel. A new cell sub-line, BCS-TC2.1, was isolated and established from these tumors. Implantation of these cells in nude mice in the absence of Matrigel-generated tumors which allowed the establishment of another tumorigenic cell sub-line, BCS-TC2.2. Matrigel and laminin, but not collagens, promote the tumorigenicity of BCS-TC2 cells, probably due to specific interactions of a pre-existing minor cell sub-population with laminin, which facilitate the initial growth of these cells in vivo. Cytogenetic analysis reveals that both sub-lines originate from the parental one, but a new marker in chromosome 9 is observed. These sub-lines present a lower degree of differentiation, as deduced from the lower CEA content, 5′-nucleotidase and alkaline-phosphatase activities. No variation is observed in the mRNA and protein expression of the 67-kDa laminin-binding protein. However, an increase in β₁ integrins and a parallel decrease in β₄ integrin were detected. Thus, the new sub-lines, compared to the parental cells, present karyotypic and phenotypic differences such as the expression of a distinctive integrin pattern. This system represents a useful model for understanding the development and progression of tumorigenicity in cancer cells. © 1996 Wiley-Liss, Inc.     

Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel.

Previously we found that the reconstituted basement membrane matrix Matrigel, when premixed with human small-cell lung carcinoma cells and injected subcutaneously into athymic mice, permitted tumor growth, whereas cells injected in the absence of Matrigel did not form tumors. In the present study, we examined additional cell types and determined some of the underlying mechanisms involved in the promotion of tumor formation by Matrigel. The tumor cell lines that we studied included transformed mouse Englebreth-Holm-Swarm tumor cells (T-EHS), human submandibular carcinoma A253 cells, mouse melanoma B16F10 cells, human epidermoid carcinoma KB cells, and human primary renal cell carcinoma cells. When coinjected subcutaneously with Matrigel, these cell lines formed rapidly proliferating tumors. Primary biopsy specimens of human colon carcinoma, when dispersed and coinjected with Matrigel, also formed tumors. Only A253, KB, and B16F10 cells formed small tumors in the absence of Martrigel, but a fivefold to tenfold increase in tumor size was observed in the presence of Matrigel. These data demonstrate a useful method for improving the growth of human tumors in athymic mice.     

Primary transfection as a mechanism for transformation of host cells by human tumor cells implanted in nude mice

Establishment of cell lines in vitro from a human lung cancer xenograft in nude mice resulted in transformed mouse cell lines. The transformed mouse cell lines expressed both mouse-specific and human-specific histocompatibility antigens. Of 3 cell lines, 2 were tumorigenic in BALB/c nude mice but not in normal mice. Tumors formed by the transformed mouse cell lines were fibroblastoid and epithelioid by histology. In addition, tumors exhibited neuroepithelial differentiation by ultrastructural and immunohistochemical analysis. Phenotypically they were similar to the original patient and human xenograft tumor. These data suggest that previous reports of host cell transformation and induction of fibrosarcomas may not be true fibrosarcomas. Human DNA sequences were present in the tumorigenic cell lines, indicating that spontaneous transfection of human tumor DNA into host cells had occurred. The implication of these findings is that human genetic information has been transferred to primary mouse host fibroblasts, which resulted in a transformed as well as a differentiated phenotype.     

Up-regulated expression of the β3 integrin and the 92-kDa gelatinase in human HT-144 melanoma cell tumors grown in nude mice

In situ changes in the repertoire of integrins and proteolytic enzymes have been demonstrated during melanoma metastasis. To investigate whether established human melanoma cell lines, injected into nude mice, could undergo phenotypic changes similar to those observed in in situ lesions, we studied 3 melanoma cell lines of distinct metastatic origin, adherent HT-144 and SK-MEL-2 cells, and non-adherent SK-MEL-1 cells for integrin expression, proteolytic enzyme repertoire and invasive potential after in vitro culture. Heterogeneity in integrin expression, such as elevated levels in ανβ in SK-MEL-1 and SK-MEL-2 cells and low expression in HT-144 cells, correlated with their in vitro invasiveness, since only the adherent HT-144 and SK-MEL-2 cells were able to invade Matrigel, and in addition, secreted a 72-kDa gelatinase. In contrast, no similar correlation could be established in nude mice, as all 3 cell lines, including the non-adherent SK-MEL-1 cells, were tumorigenic when injected s.c., while only HT-144 consistently produced experimental lung metastasis. Immunochemical analysis of the integrin profile in s.c. xenografts revealed over-expression of αν, β1, and β3 integrins exclusively in HT-144 cells, as well as increased expression of β3 in HT-144 cell lung metastases, as confirmed by PCR analysis using species-specific primers, while zymography and Western-blot analysis demonstrated de novo expression of the 92-kDa gelatinase MMP-9 in HT-144 xenografts. Our results highlight a positive correlation between up-regulated β3 integrin and MMP-9 expression in human HT- 144 melanoma cell tumors grown in nude mice. © 1996 Wiley-Liss, Inc.