Ezrin interacts with cortactin to form podosomal rosettes in pancreatic cancer cells.

BACKGROUND AND AIMS: Pancreatic cancer is a highly invasive malignancy. Ezrin, a plasma membrane-cytoskeletal linker protein, is associated with the invasive behaviour of cancers. The purpose of this study was to elucidate a possible molecular mechanism for the invasive phenotype. METHODS: Using a combination of techniques, such as western blotting, co-immunoprecipitation, confocal and light microscopy, invasion and adhesion assays, organotypic cultures and human samples as well as RNA interference (RNAi) and expression of various mutant ezrin constructs, the dynamic molecular nature of podosomes in pancreatic cancer was dissected out. RESULTS: Podosome and podosomal rosette formation in pancreatic carcinoma (PaCa3) cells is ezrin dependent and associated with adhesion to fibronectin with subsequent digestion of this substrate. Ezrin binds to increasing amounts of cortactin during formation of the podosomal rosette, with the C-terminal region, specifically the actin-binding domain, mediating this molecular linkage. Further, it is shown that phosphorylation of Tyr353 and Thr567 sites on ezrin (conventionally shown to translocate ezrin to the plasma membrane) is not required for podosome formation. The podosomal rosette is revealed to be a highly dynamic and transient structure, which can metamorphose into other cellular processes, such as filopodia or lamellipodia, and thereby enable epithelial cancer cells to "palpate" the underlying substrate and modify their cytoskeletal behaviour accordingly. In human tumour tissues and organotypic cultures, specific subcellular expression of ezrin (basal membranous; cellular processes invading stroma) in pancreatic cancer cells can be correlated with tumour progression and disease-free survival (log-rank test (Mantel-Cox), p = 0.019). CONCLUSION: Podosomes and their rosettes are driven by ezrin-cortactin interaction and this plays a role in pancreatic cancer invasion.

Biological diversity in metastatic neoplasms: origins and implications.

Whether neoplasms are unicellular or multicellular in their origin, the process of tumor evolution and progression can rapidly generate biological diversity. Metastases result from the survival and proliferation of specialized subpopulations of cells within the parent tumor. Metastases may have a clonal origin and different metastases may develop from different progenitor cells. However, as with the primary tumor, the origin of metastases is unimportant since the process of tumor evolution and progression can generate biological diversity within and among different metastatic foci.

Effect of cytoskeleton-disrupting agents on the metastatic behavior of melanoma cells.

The effects of treatment with colchicine, cytochalasin B, and low temperature (4 degrees C) on the metastatic behavior of the B16-F10 melanoma cell line were examined. The growth of metastases and the distribution of radiolabeled tumor cells were monitored in inbred C57/BL6 mice given iv injections of B16-F10 cells. Cells treated previously with both drugs, but not with low temperature, produced fewer lung nodules than did control cells and displayed alterations in tumor dissemination patterns. In vitro studies revealed that both drugs reduced the rate of adhesion of the tumor cells to bovine endothelial cell monolayers, the rate of migration from agarose droplets, the formation of homotypic aggregates, and agglutination by wheat germ agglutinin. The drugs also induced morphologic alterations of the cells grown in monolayer culture but had little effect on cell volume.

Correlation of tumor growth inhibitory activity of macrophages exposed to adriamycin and adriamycin sensitivity of the target tumor cells.

Variant cell lines of the murine fibrosarcoma UV-2237, selected for doxorubicin [adriamycin (ADM)] resistance, were used to study the tumoricidal activity of macrophages that had been exposed to ADM. Free ADM (0.01-1 microgram/ml) was cytotoxic to the sensitive UV-2237M parent and to the partially sensitive UV-2237M-revertant cell lines, whereas the ADM-resistant UV-2237M ADMR line was unaffected by these levels of ADM. Macrophages harvested from the peritoneal cavities of mice given ip injections of 10 mg ADM/kg body weight 1 day or 4 days previously inhibited proliferation of the 3 cell lines, an effect that was directly correlated to the degree of ADM sensitivity of each cell line. Macrophages exposed in vitro to ADM (from 0.01 to 1 microgram/ml) inhibited the growth of, and eventually were cytolytic to, the parent and the revertant cell lines; these ADM-exposed macrophages did not affect the UV-2237M-ADMR cell line. The correlation between the antitumor effects of macrophages exposed to ADM and the ADM susceptibility of tumor cells suggests that ADM-exposed macrophages exert their effect by the release or transfer of the stored drug.

A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice.

The ability of the human A375 melanoma cell line to produce experimental and spontaneous in young BALB/c nude mice was examined. Cloned lines, obtained by isolation in semisolid agar, differed significantly (4/10 cloned lines examined, P less than or equal to .005) from the parent tumor line with regard to their ability to form lung tumor nodules subsequent to iv injection. Lines established from such lung tumor nodules were more metastatic than the parent line following both iv and sc injection. These results show that the human melanoma cell line used in these studies contained cells with diverse metastatic potential and suggest that metastasis in the nude mouse results from the preferential selection of metastatic subpopulations.

Expression of collagenase IV (basement membrane collagenase) activity in murine tumor cell hybrids that differ in metastatic potential.

Expression of a basement membrane collagen-degrading metalloprotease activity (collagenase IV) was studied in a series of murine cell hybrids derived from fusions between highly metastatic cells (B16-F10RR) or moderately metastatic cells (UV-2237RR) and tumorigenic cells (K-1735 clone 16) or normal cells [peritoneal macrophages (PEC) or C3H mouse embryo fibroblasts (C3H-F)]. The collagenase IV activity of the parent cells and the hybrids was assayed in vitro and compared to the metastatic propensity of the same cells evaluated in both syngeneic (C57BL/6 X C3H/HeN)F1 mice and BALB/c nude mice. The level of collagenase IV activity secreted by the parent lines correlated with their metastatic capacity. The highly metastatic B16-F10RR line secreted the highest enzyme activity, whereas the tumorigenic but nonmetastatic K-1735 clone 16 and the normal parents PEC and C3H-F secreted the lowest enzyme activity. The enzyme activity was completely inhibited with EDTA. The hybrid derived from fusion of cells from two metastatic cell lines as well as hybrids derived from a metastatic and a nonmetastatic tumor cell line expressed higher levels of collagenase IV activity than either parent, and this expression was associated with a high ability to produce metastases in both nude and syngeneic mice. Fusion of metastatic cells with normal cells produced hybrid cells that exhibited suppression of both collagenase IV activity and metastatic capacity. Collagenase IV activity and metastatic propensity can, therefore, be altered by somatic cell hybridization; in the series of hybrids examined in these experiments the expression of type IV collagen-degrading metalloprotease activity and the metastatic ability were closely correlated, which suggests that collagenase IV activity and other properties required for metastasis are genetically linked.

Use of tissue-specific expression of the herpes simplex virus thymidine kinase gene to inhibit growth of established murine melanomas following direct intratumoral injection of DNA.

We report here the use of the 5' flanking region of the murine tyrosinase gene to direct expression of the herpes simplex virus thymidine kinase (tk) gene specifically to murine melanoma cells, whilst not permitting expression in a range of other cell types. Expression of the herpes simplex virus tk gene from the tyrosinase promoter in melanoma cells rendered them sensitive to killing by ganciclovir (100% cell death of a tk-expressing B16 clone after 12 days in culture at 1 microgram/ml ganciclovir). We also observed a substantial bystander killing effect when expressing cells were mixed with nontransfected parental B16 cells. When transfected murine melanoma cells expressing tk were injected into syngeneic mice both their tumorigenicity and experimental metastatic potential were abrogated completely when the mice were treated with ganciclovir (27 of 28 mice treated with water developed progressively growing tumors versus 1 of 30 in the ganciclovir-treated group). Direct injection of the tk gene under control of the tyrosinase promoter into established tumors in mice, followed by treatment with ganciclovir, led to significant reductions in resultant tumor size relative to the size of tumor developing in mice treated with water (median tumor weight, 1.65 g versus 2.75 g). Therefore, direct transfer of recombinant genes by injection of DNA can significantly reduce established tumor burden in vivo.

In vitro and in vivo targeting of gene expression to melanoma cells.

Gene therapy protocols for cancer usually involve removal of tumor cells, culture in vitro to allow gene transfer, and subsequent reintroduction in vivo. Targeting therapeutic genes to tumor cells in situ requires an accuracy of gene delivery that currently is not possible with the use of existing techniques. To overcome these limitations we have used two promoters, which are preferentially active in melanocytic cells, to direct gene expression specifically to melanoma cells both in vitro and in vivo. Here we describe experiments showing that as little as 769 base pairs of the 5'-flanking regions of the tyrosinase, and 1.4 kilobase pair of the tyrosinase-related protein 1, genes are sufficient to direct expression of the beta-galactosidase gene to both human and murine melanoma cells and melanocytes, while not permitting expression in a range of other cell types in vitro. These promoters showed high levels of activity in 12 of 14 murine and human melanoma cell lines tested but showed only basal levels of activity, similar to that of a promoterless construct, in a range of 12 other cell types. Cell type specificity is maintained when the construct is delivered to cells either by physical means or by inclusion of the cell type-specific expression cassette into a retroviral vector. Direct injection of DNA, encoding the beta-galactosidase gene expressed from either promoter, into established B16 melanomas or Colo 26 tumors in syngeneic mice resulted in extensive transduction of tumor cells in the B16 melanomas (approximately 10% of tumor cells expressing 10 days after DNA injection), whereas no blue-staining cells were seen in the Colo 26 tumors. The reporter gene was expressed in melanoma cells and in some normal melanocytes but not in other surrounding normal tissue. We propose that the combination of a tissue-specific promoter driving a therapeutic gene, with delivery of such a construct directly to sites of tumor growth in vivo, either by direct DNA injection or by retroviral infection, may provide significantly enhanced safety for gene therapy for solid tumors.

Inhibited growth of a reticulum cell sarcoma (M5076) induced in vitro and in vivo by macrophage-activating agents.

The tumor M5076 is highly malignant in vivo; however, in vitro M5076 tumor cells express many of the differentiated characteristics of an activated macrophage. Recently, we reported that this macrophage tumor (M5076) can be induced to cease cellular division following in vitro exposure to macrophage-activating agents, which we felt was due to the induction of terminal differentiation. In the present study, we report that not all macrophage-activating agents halt the proliferation of M5076 tumor cells in vitro, and we present evidence that the treatment of mice bearing M5076 tumor cells with lipopolysaccharide is a highly effective therapeutic modality. Therapy protocols using multiple injections of lipopolysaccharide are capable of prolonging the survival and reducing the metastatic tumor burden of mice with a large tumor burden at the onset of therapy. This indicates that caution should be exercised in the use of M5076 tumors as a test model for chemotherapeutic agents, since the slightest contamination of an experimental drug with lipopolysaccharide would result in spurious positive results.

Role of organ selectivity in the determination of metastatic patterns of B16 melanoma.

The preferential growth of B16 melanoma metastases in specific organs was studied. Following the i.v. injection of B16 melanoma cells into syngeneic C57BL/6 mice, tumor growths developed in the in situ lungs and in grafts of pulmonary or ovarian tissue implanted either s.c. or i.m. In contrast, neoplastic lesions failed to develop in control grafts of similarly implanted renal tissue or at the site of a surgical trauma. Parabiosis experiments suggested that the growth of the B16 melanoma in ectopic lung or ovary tissue was due to the immediate arrest of circulating neoplastic cells and not to shedding of malignant cells from foci growing in the in situ lungs. Quantitative analysis of tumor cell arrest and distribution using cells labeled with [125I]-5-iodo-2'-deoxyuridine indicated that the growth of tumors in the implanted organs was not due to an enhanced initial arrest of B16 cells. No significant differences in immediate tumor cell arrest were detected between implanted fragments of lungs (tumor positive) and kidney (tumor negative) or between organ-bearing and contralateral control limbs. We conclude that the outcome of metastasis is dependent on both tumor cell properties and host factors. This conclusion supports the "seed and soil" hypothesis to explain the nonrandom pattern of cancer metastasis.

Biological and experimental consequences of the zonal composition of solid tumors.

The K-1735 melanoma is a transplantable murine tumor which, when growing s.c., is composed of melanotic and amelanotic areas. Results from trocar transplantation studies of tumor fragments show that expression of this phenotypic diversity can be terminated within very few passages. In parallel experiments, passage of cell suspensions derived from the whole tumor maintains this heterogeneity of melanin production. The implications of these findings are discussed.

An in vitro quantitative assay for tumor cell invasion.

An in vitro quantitative assay of tumor cell invasion is described. The assay measures the rate at which [125I]iododeoxyuridine-labeled tumor cells migrate through the chorioallantoic membrane of developing chicken embryos. Invasion chambers were prepared from amber latex cylinders, chorioallantoic membrane, and thread ligatures. The chambers were placed in glass vials to rest on discs of photographic sponge immersed in tissue culture medium. Labeled tumor cells were added to the chamber, and the vial was incubated at 37 degrees. At various time points individual chambers were monitored to determine the number of viable, labeled cells that had passed through the membrane and were present in the medium, on the glass vial, or in the sponge. The application of the assay may be of use in delineating the mechanisms of tumor cell invasion.

Factors influencing blood supply in wound granuloma quantitated by a new in vivo technique.

A new quantitative assay for measuring angiogenesis in a s.c. located sponge implant in rats is described. Using this model, which detects neovascularization by measuring alterations in 133Xe clearance, it has been shown that the known angiogenic factors, transforming growth factor alpha and tumor necrosis factor alpha, cause maximum vascularization of the sponge to occur by Day 11 postimplantation compared with Days 15 to 17 in control animals. The monokine interleukin 1 alpha is shown to be strongly angiogenic, suggesting that more than one macrophage-derived cytokine may be the active mediator in macrophage-induced angiogenesis. Extracellular matrix proteins appear to play a role in regulating the angiogenic response such that presoaking sponges in laminin (40 micrograms/ml) or fibrinogen (500 micrograms/ml) solutions induced a significant reduction in the time taken to achieve maximum 133Xe clearance values; no such enhancement of neovascularization was observed when sponges were presoaked in type IV collagen (100 micrograms/ml) solution. The assay described here, which is reproducible, objective, and quantitative, should be of considerable use in elucidating the molecular basis of angiogenesis regulation.

Induction of tumorigenicity and lack of in vitro growth requirement for 12-O-tetradecanoylphorbol-13-acetate by transfection of murine melanocytes with v-Ha-ras.

A nontumorigenic line of murine melanocytes, Mel-ab, has been transfected with the v-Ha-ras gene under transcriptional control of the Moloney murine leukemia virus long terminal repeat. Transfectants produced rapidly growing undifferentiated melanomas in recipient mice. The inhibition of melanin production in transformed cells, observable both in vitro and in vivo, suggests that ras may affect melanocyte cytodifferentiation. Mel-ab cells require the continual presence of 12-O-tetradecanoylphorbol-13-acetate, or other activators of protein kinase C, for in vitro growth. Transfectants expressing v-Ha-ras no longer manifested this requirement and were actually growth inhibited by the addition of protein kinase C activators. These results are consistent with the notion that ras acts via the protein kinase C pathway in conferring autonomous growth on Mel-ab cells.

Metastatic behavior of an adriamycin-resistant murine tumor.

The metastatic behavior of a murine fibrosarcoma (UV-2237M-ADMR) resistant to Adriamycin (ADM) (doxorubicin) was investigated. Subclones isolated from the UV-2237M-ADMR line, which originated from a single colony, generally displayed a similar degree of resistance to ADM (eight out of nine clones did not differ from this UV-2237M-ADMR line). In contrast, they differed significantly in their capacity to form lung colonies after i.v. injection (six of nine clones differed significantly from the UV-2237M-ADMR line, p less than or equal to 0.005, Mann-Whitney U test). The UV-2237M-ADMR cell line maintained resistance to ADM even after 17 weeks of growth in syngeneic mice, although a gradual decrease in resistance was observed over this time. Spontaneous metastases from the UV-2237M-ADMR tumor commonly retained resistance to ADM. Of 18 cell lines, each established from an individual lung nodule, 16 showed plating efficiencies in the presence of ADM comparable to that of the primary UV-2237M-ADMR tumor. The remaining two lines had partially reverted to the sensitive state. The i.v. administration of ADM significantly reduced the lung tumor burden of mice with ADM-sensitive UV-2237M tumors but failed to affect the lung tumor burden of mice with UV-2237M-ADMR tumors. The UV-2237M-ADMR tumor line, exhibiting as it does both drug-resistant and metastatic behavior, provides a useful model system with which to investigate the metastatic process and the development of drug resistance.

Isolation and preliminary characterization of an Adriamycin-resistant murine fibrosarcoma cell line.

A variant cell line (UV-2237-ADMR) resistant to the anthracycline antibiotic Adriamycin (Doxorubicin) was selected in vitro from the murine UV-2237 fibrosarcoma tumor cell line. Resistance to Adriamycin proved to be a stable characteristic of the UV-2237-ADMR line, whether the line was grown in vivo or in vitro. The UV-2237-ADMR line also exhibited increased resistance to N-trifluoroacetyladriamycin-24-valerate, daunorubicin, actinomycin D, amsacrine, mitomycin C, vinblastine, and vincristine but not to bleomycin. Cell-cell hybridization studies showed that the Adriamycin resistance is an incompletely dominant trait. Uptake and efflux studies with [14C]Adriamycin indicated that the resistance exhibited by the UV-2237-ADMR line was due to both reduced uptake of the drug and an increased active efflux.

Characterization of a murine ovarian reticulum cell sarcoma of histiocytic origin.

We have studied the M5076 tumor, a transplantable murine reticulum cell sarcoma that arose spontaneously in the ovary of a C57BL/6 mouse. This tumor displays functional and ultrastructural characteristics indicating that it is of macrophage origin. Cells from the M5076 tumor are phagocytic, form rosettes with sheep red blood cells, mediate antibody-dependent cellular cytotoxicity against 51Cr-labeled red blood cells, and display macrophage-like cytotoxicity against syngeneic tumor target cells but do not exhibit any natural killer cell activity. The tumor cells possess lysozyme, nonspecific esterase, and phosphatase activities comparable to that seen in rodent macrophages. Ultrastructural examination revealed phagocytic vacuoles and a lack of tight junctions typical of macrophage morphology. Karyotype analysis showed that M5076 tumor cells are hypodiploid with a high percentage (greater than 80%) of metacentric chromosomes that serve as an excellent marker for identification of these tumor cells.

Metastatic behavior of a murine reticulum cell sarcoma exhibiting organ-specific growth.

The metastatic properties of the M5076 tumor, a reticulum cell sarcoma of ovarian origin, were examined. This tumor metastasizes preferentially to the peritoneal viscera (liver, ovaries, spleen, and kidneys) regardless of the site or route of tumor cell injection. Subcutaneous tumor growth followed by direct invasion into the peritoneum resulted in extensive generalized peritoneal involvement. However, when tumor cells were injected in the dorsum, external ear, or footpad, fewer and primarily hepatic metastases developed. Hepatic, splenic, ovarian, and renal tumor colonies were formed after i.v. injection of tumor cells. Radiolabeled tumor cells were used to study the arrest, distribution, and survival of tumor cells injected i.v. These tumor cells were rapidly arrested in the lungs and were retained there for 3 to 4 days. They then slowly detached, recirculated, and were arrested in the liver, where they subsequently developed into tumor nodules. These results strongly support the "soil-seed" hypothesis of metastatic growth and demonstrate that long-term retention of tumor cells in an organ need not result in the formation of a clinically obvious tumor nodule.

Comparative studies on the quantitative analysis of experimental metastatic capacity.

The purpose of these studies was to establish a procedure for determining the relative experimental metastatic potential of unrelated murine tumors. We used three tumors (the B16-F10 melanoma, which is syngeneic to the C57BL/6N mouse, and the K-1735 melanoma and the UV-2237 fibrosarcoma, which are syngeneic to the C3H/HeN mouse). Various numbers of tumor cells were injected into normal or immunosuppressed syngeneic recipients and into 3-week-old BALB/c nude mice. At appropriate intervals, the recipient mice were killed, and the metastatic burden was determined. The number of experimental metastases was not linearly correlated with cell input. Thus, simply comparing the incidence of metastasis resulting from the injection of one predetermined dose of tumor cells did not allow for determination of their relative metastatic capacities. More reproducible and meaningful results were obtained by introducing increasing numbers of viable tumor cells admixed with a constant number of nontumorigenic (X-irradiated) tumor cells serving as carrier. The incidence of metastasis by few or many injected cells is influenced by host factors such as immune status, and therefore determinations of the true metastatic nature of any given tumor necessitate the choice of an appropriate recipient.

Inhibition of cellular division of a murine macrophage tumor by macrophage-activating agents.

Although the murine reticulum cell sarcoma M5076 is highly malignant in vivo, in vitro it displays many of the functional characteristics of an activated macrophage, such as phagocytosis and tumor cytotoxicity. This study was designed to determine what effect macrophage-activating agents would have on the function and growth of M5076 cells. Exposure of M5076 tumor cells to substances that activate normal macrophages to the tumoricidal state rapidly and irreversibly induced cessation of cellular division. The treated tumor cells, however, retained the same characteristics as those of untreated M5076 cells in vitro with respect to viability and the activated macrophage functions of phagocytosis and tumor cytotoxicity. Even after a short exposure to lipopolysaccharide, the ability of M5076 cells, injected i.v. into syngeneic mice, to form tumor nodules was greatly reduced. These results indicate that a highly malignant tumor of macrophage origin, M5076, can be induced to cease cellular division while retaining the functional attributes of an activated macrophage. We speculate that the exposure of M5076 to macrophage-activating agents results in the induction of terminal differentiation of this tumor.