The structure of invadopodia in a complex 3D environment.

Invadopodia and podosomes have been intensively studied because of their involvement in the degradation of extracellular matrix. As both structures have been studied mostly on thin matrices, their commonly reported shapes and characteristics may differ from those in vivo. To assess the morphology of invadopodia in a complex 3D environment, we observed invadopodial formation in cells grown on a dense matrix based on cell-free dermis. We have found that invadopodia differ in morphology when cells grown on the dermis-based matrix and thin substrates are compared. The cells grown on the dermis-based matrix display invadopodia which are formed by a thick protruding base rich in F-actin, phospho-paxillin, phospho-cortactin and phosphotyrosine signal, from which numerous thin filaments protrude into the matrix. The protruding filaments are composed of an F-actin core and are free of phospho-paxillin and phospho-cortactin but capped by phosphotyrosine signal. Furthermore, we found that a matrix-degrading activity is localized to the base of invadopodia and not along the matrix-penetrating protrusions. Our description of invadopodial structures on a dermis-based matrix should greatly aid the development of new criteria for the identification of invadopodia in vivo, and opens up the possibility of studying the invadopodia-related signaling in a more physiological environment.

[Diethylstilbestrol-induced uterine sarcoma in mice]. / Sarkomy matki myshei, indutsirovannye diétilstil'béstrolom.

Female mice of CBA strain received diethylstilbestrol (DES) 1 g body weight intraperitoneally. High incidence of histiocytic uterine sarcoma was observed in parents (25%), first generation (F1), descendants (10.9%), and generation F2m (through F1 males mated with females in control) (17.8%). Morphologically, tumor cells examined through light and electron microscopy were referred to as histiocyte-like elements. Half of the animals had metastases in the liver, kidneys, lungs, spleen, pineal and adrenal glands and stomach. The development of tumors in generation F2m, which was not exposed to DES, might be accounted for by "transgeneration" carcinogenesis, i.e. passage of carcinogenic effect through a generation.

Spontaneously formed tumorigenic hybrids of Meth A sarcoma cells and macrophages in vivo.

We have recently demonstrated that malignant cells can hybridize with tissue macrophages in vitro, giving rise to tumorigenic hybrids. We now demonstrate that this can occur spontaneously in vivo as a result of fusion between inoculated Meth A sarcoma cells and host cells, presumably macrophages. Thus, from tumor cell suspensions prepared by collagenase perfusion and density centrifugation, hybrid cells could be isolated that were neoplastic but in contrast to Meth A expressed macrophage markers and had phagocytic capacity. Their morphologic features were intermediate between Meth A and macrophages. By taking advantage of a semiallogeneic experimental system by inoculation of Meth A cells from BALB/c (H-2 K(d)) into (BALB.K x BALB/c) F(1) (H-2(k/d)), hybrid cells from these tumors could be shown to express MHC antigens of both the Meth A and the host haplotypes. Hybrid cells grew faster than Meth A cells in vivo, indicating acquisition of growth-promoting properties through heterotypic cell fusion.

Extremely low frequency (ELF) pulsed-gradient magnetic fields inhibit malignant tumour growth at different biological levels.

Extremely low frequency (ELF) pulsed-gradient magnetic field (with the maximum intensity of 0.6-2.0 T, gradient of 10-100 T.M(-1), pulse width of 20-200 ms and frequency of 0.16-1.34 Hz treatment of mice can inhibit murine malignant tumour growth, as seen from analyses at different hierarchical levels, from organism, organ, to tissue, and down to cell and macromolecules. Such magnetic fields induce apoptosis of cancer cells, and arrest neoangiogenesis, preventing a supply developing to the tumour. The growth of sarcomas might be amenable to such new method of treatment.

Spontaneous hybridization of macrophages and Meth A sarcoma cells.

We present evidence of hybridization between Meth A sarcoma cells and syngeneic as well as semigeneic peritoneal macrophages. The resultant hybrids are characterized by morphology, membrane markers, ploidy, chromosomal content and functional features. Briefly, after a few days of coculture, cells appeared with morphology intermediate between the 2 original cell types. Typical macrophage surface molecules appeared in the hybrids. Meth A cells were labeled with red fluorescence and macrophages with green fluorescence. After 4 days in vitro, hybrids with yellow fluorescence appeared. Macrophages from BALB.K mice (H-2 K(k)) were cocultivated with Meth A cells from BALB/c mice (H-2 K(d)). The semigeneic hybrids displayed both specificities, as demonstrated by flow cytometry. The hybrids appeared moderately phagocytic, less so than the macrophages and markedly more so than the essentially nonphagocytic Meth A cells. The hybrids had a mean number of 76 chromosomes, as opposed to 53 in the Meth A cells and 40 in the macrophages. The macrophage DNA index was set at 1; Meth A cells were found to have an index of 1.6 in G1 phase, and the hybrids had a 2.6 index. The hybrids grew more slowly in vitro than Meth A cells, but grew faster in vivo.

Evidence for characteristic vascular patterns in solid tumours: quantitative studies using corrosion casts.

The vascular architecture of four different tumour cell lines (CaX, CaNT, SaS, HEC-1B) transplanted subcutaneously in mice was examined by means of microvascular corrosion casting in order to determine whether there is a characteristic vascular pattern for different tumour types and whether it differs significantly from two normal tissues, muscle and gut. Three-dimensional reconstructed scanning electron microscope images were used for quantitative measurements. Vessel diameters, intervessel and interbranch distances showed large differences between tumour types, whereas the branching angles were similar. In all tumours, the variability of the vessel diameters was significantly higher than in normal tissue. The quantitative data provide strong evidence for a characteristic vascular network determined by the tumour cells themselves.

Morphological and biological characterization of two mesenchymal murine tumors and the modulation of their growth parameters by n-3 and n-6 polyunsaturated fatty acids.

Certain tumor growth parameters (GP) of two mesenchymal transplantable tumors maintained on C57BL/6J mice were characterized. Considering that many experimental, clinical and epidemiologic data have indicated that n-3 and n-6 essential fatty acids are nutrients which may delay the development as well as improve the course of cancer, GPs were evaluated on hosts fed on a semisynthetic formula containing 5% of corn oil (CO) or cod liver oil (CLO) and stock diet (C group). Although survival and latency time of tumor-bearing mice were shortened, other GP as percentage of successful implants were improved by both oils in sarcoma-bearing hosts, suggesting that n-3 and n-6 fatty acids might play a modulating role for the development of these tumors.

Lewis lung carcinoma and two transplantable sarcomas in mice as experimental therapy models: biological characteristics and cell population kinetics.

The biological characteristics of three transplantable tumours: two sarcomas (SaL, MCA) and Lewis lung carcinoma (LLC) have been studied. We investigated histology, DNA ploidy and cell kinetics parameters of the tumours. All examined tumours were aneuploid and rapidly proliferating, with the hyperdiploid fraction greater than 60%. The SaL tumour was found to have the lowest mean aneuploid bromodeoxyuridine labelling index (LI) equal to 21%, while the highest LI of 35.8% was measured for the LLC tumour. The mean S-phase time was short, lasting 8.5 - 10.9 hrs. The potential doubling time (T(pot)) assessed by BrdUrd staining and flow cytometry were as follows: 37.1 hours for SaL, 22.7 hours for MCA and 21.4 hours for LLC tumour. The MCA had the shortest volume doubling time (T(d)) equal to 1.7 days and the longest one, equal to 4.7 days, was found for the LLC. The lowest cell loss was found in the MCA tumour (44%), while the highest in the LLC tumour (81%). As all the examined tumours proliferate rapidly, there is a capacity for accelerated repopulation and therefore the tumours seem to be good models for experimental radiotherapy.

Macrophage cytotoxicity against murine meth A sarcoma involves nitric oxide-mediated apoptosis.

We have studied the cytotoxic effect of stimulated macrophages on Meth A tumor cells in vitro. When stimulated with interferon-gamma and soluble beta-1,3-D-glucan, macrophages exerted cytotoxicity towards syngeneic Meth A tumor cells. This cytotoxicity was associated with a high level of nitric oxide production. Both cell death and nitric oxide production were significantly inhibited by the addition of aminoguanidine, a specific inhibitor of inducible nitric oxide synthase (iNOS), to the culture medium. The cytotoxic effect was accompanied by internucleosomal cleavage of DNA as shown by electrophoresis and DNA fragmentation assay.

Variability of differentiation patterns in xenotransplanted spindle cell sarcomas: a histomorphological, immunohistochemical, and ultrastructural study.

Three leiomyosarcomas, 3 nerve sheath sarcomas, 1 rhabdomyosarcoma, and 1 sarcoma not otherwise classifiable with 17 of their xenografts, grown on nude mice, were analyzed to assess the degree of concordance between histomorphology, immunohistochemistry, and ultrastructure in spindle cell sarcoma xenograft differentiation. Histomorphology was inconclusive or misleading in 4/8 sarcoma strains and immunohistochemistry in 4/8 originals and in 10/17 xenografts, although specific patterns had been identified ultrastructurally. Electron microscopy was superior to immunohistochemistry and histomorphology in spindle cell sarcoma differential diagnosis. A further purpose of this study was to clarify whether spindle cell sarcoma xenografts retain the morphological characteristics of their primaries. Histomorphological features of the primaries were preserved over all passages, whereas the immunohistochemical marker profiles as well as the ultrastructural phenotypes changed in 14/17 xenografts and in 8/17 xenografts, respectively. Moreover, unusual bidirectional or tridirectional patterns of differentiation were identified ultrastructurally with leiomyomatous as well as Schwann cells occurring side by side and with MFH-like areas in 5/17 xenotransplants. These findings suggest genetic instability of tumor cells and may be important in the consideration of mesenchymal differentiation pathways.

Video rate confocal laser scanning reflection microscopy in the investigation of normal and neoplastic living cell dynamics.

The introduction of video rate confocal laser scanning microscopes (VRCLSM) used in reflection mode with high magnification, high aperture objective lenses and with further magnification by a zoom facility allowed the first detailed observations of the activity of living cytoplasm and offered a new tool for investigation of the structural transition from the living state to the specimen fixed for electron microscopy (EM). We used a Noran Odyssey VRCLSM in reflection (backscattered) mode. A greater degree of oversampling and more comfortable viewing of the liver or taped video image was achieved at zoom factor 5, giving a display monitor field width of 10 microns. A series of mesenchyme derived cell lines--from normal cells to sarcoma cells of different malignancy--was used to compare behaviour of the observed intracellular structures and results of fixation. We contrasted the dynamic behaviour of fine features in the cytoplasm of normal and neoplastic living cells and changes induced by various treatments. The tubulomembraneous 3D structure of cytoplasm in living cells is dynamic with motion observable at the new limits of resolution provided by VRCLSM. All organelles appear integrated into one functional compartment supporting the continuous 3D trafficking of small particles (vesicles). This integrated dynamic spatial network (IDSN) was found to be largest in neoplastic cells.

A simple and accurate method for 3-D measurements in microcorrosion casts illustrated with tumour vascularization.

Microvascular corrosion casting is an established method to investigate vascular patterns of nearly all organs. Trying to evaluate these specimens quantitatively using native scanning electron microscope (SEM) pictures always implied a big error, because the information of depth cannot be taken into account. However, SEM stereo pairs allow for exact measurements. Tumour microcorrosion casts were used to demonstrate the feasibility of this 3-D quantification method. The information of depth was calculated for each measuring point using the parallax. From the resulting coordinates the intervascular distances, the interbranching distances as well as the interbranching angles were determined. We found significant differences between all investigated tumours. Reproducibility tests and tests for the greatest error possibilities resulted in a maximum deviation of 2.5% to be expected. Consequently this method is suitable for all application ranges of microvascular corrosion casting as a quantitative determination method.

Expression of manganese superoxide dismutase reduces tumor control radiation dose: gene-radiotherapy.

This study investigated the in vitro and in vivo radiation response of tumor cells transfected with human manganese superoxide dismutase (MnSOD) cDNA. A major objective was to test the potential tumor suppressive effect of MnSOD in vivo. Tumor cells studied were an in vitro line derived from a murine spontaneous fibrosarcoma, FSa-II, which expressed an undetectable MnSOD activity. These cells were transfected with pSV2-NEO plasmid (NEO line) or cotransfected with MnSOD plasmid plus pSV2-NEO plasmid (SOD lines) as described previously. The cell lines used were SOD-L and SOD-H, which expressed, respectively, low and high MnSOD activities after transfection, and NEO and parental FSa-II controls. Both SOD-L and SOD-H cell lines were slightly more resistant to ionizing radiation than were the two control cell lines when irradiated in vitro in the presence of oxygen. The dose-modifying factors calculated at the survival level of 0.01 were 1.13 and 1.15 for the SOD-L and SOD-H cells, respectively. To investigate potential tumor suppressive effects, animal tumors of 4 mm diameter were irradiated in vivo under hypoxic conditions, and the radiation dose to control one-half of the irradiated tumors (TCD50) was determined for each tumor. The TCD50S obtained on the basis of the tumor control rate in 120 days after irradiation were substantially lower for the SOD-H and SOD-L tumors compared to the NEO tumors. They were 22.9, 28.6, and 47.5 Gy for SOD-H, SOD-L and NEO tumors, respectively. To analyze these data, survival curves were obtained for hypoxic cells by irradiating NEO and SOD-H tumors under hypoxic conditions in vivo and assaying in vitro. Analysis of these curves suggests that the decrease in the TCD50S of SOD tumors is attributable to the reduced tumorigenicity in these tumors. The hypoxic cell survival curves also showed that SOD did not protect cells from radiation in the absence of oxygen. Electron microscopy showed no morphological differences between these cells. These results suggest that the fraction of tumorigenic cells could be reduced by expression of MnSOD, resulting in a substantial decrease in the TCD50.

Fiber matrix descriptors from permeability data without requiring membrane thickness: theory, results, and optimization.

OBJECTIVE: Permeability of basement membrane and all other barriers contains a term for membrane thickness (delta x). This naturally leads to development of methods for measuring delta x that are imprecise, inaccurate, expensive, subject to preparation artifact, and inattentive to variability. Although height and shape of permeability (P) vs. probe radius (aE) curves are sensitive to delta x, In(P) or In (P/free diffusivity or D0) curves have shapes independent of delta x. It should, thus, be possible using such characteristics to determine fiber radius (rf) and void volume ratio (epsilon) without delta x. We developed such a method to derive membrane structure by the standard model of Ogston and present its experimental evaluation. METHODS: Basement membranes were self-assembled using 1:1 Matrigel: 0.01 M Tris/150 mM NaCl/1.0 mM CaCl2 buffer on 0.4-mu polycarbonate supports with transport measured in diffusion chambers using FITC-labeled hydroxyethyl starch probes from 25 to 102 A in radius. Sampling was at 0.5 hr and then for each hour up to 5. Other membranes were measured 7 days after formation. RESULTS: The best fit of the new technique occurred at 3 hr with R2 = 0.949 +/- 0.003 SEM, rf = 36.8 = 2.4 A, and epsilon = 0.87 +/- 0.02. Membranes studied for 7 days showed more variability but essentially the same characteristics. CONCLUSIONS: Membrane thickness is not necessary to reduce permeability of basement membrane to structure, and optimum sampling time is 3 hr.

Effects of vinblastine on cell membrane fluidity and the growth of SA-1 tumor in mice.

Cell membranes can be targets of some anti-cancer drugs. Therefore, the purpose of this study was to determine whether vinblastine (VLB) can also affect the tumor cell membrane. On the in vivo SA-1 tumor model, alteration of cell membrane fluidity (measured by electron paramagnetic resonance, EPR), cytotoxicity and morphological changes of the SA-1 tumor cells after VLB treatment were studied. The cytotoxic effect of VLB was biphasic, with an initial fast increase in cytotoxicity followed by a plateau. The surviving cells had increased membrane fluidity and were morphologically changed. The dose-response curve of VLB on membrane fluidity was also biphasic with an initial fast increase in membrane fluidity followed by a plateau. Since dose-response curves of VLB cytotoxicity and its effect on membrane fluidity were similar, there was a high correlation between both effects. The effect of VLB on membrane fluidity was the most pronounced at 24 h and 48 h after treatment. The results of this study indicate that VLB affects cell membrane by increasing the membrane fluidity of SA-1 tumor cells in vivo in a dose-and time-dependent manner. Therefore, this finding may be beneficially implemented also in priming cells for other cytotoxic drugs and for appropriate timing of drug sequence in combined schedules.

A temporal study of the lesions induced by MoMuSV-349.

We used time point studies to document the progression of neoplasms, haematologic abnormalities and associated lesions induced by Moloney murine sarcoma virus-349 (MoMuSV-349). BALB/c mice inoculated intraperitoneally with MoMuSV-349 first developed histologically discernible lesions at 14 days post-inoculation (d.p.i.). The initial neoplasms were characterized by whorls of fusiform or spindle-shaped cells enmeshing dense infiltrates of neutrophils and macrophages. By 21 d.p.i., clinical signs associated with MoMuSV-349 infection were evident. The distribution of the neoplasms was more widespread, although the histologic appearance of the tumours was very similar to that found at 14 d.p.i. All mice sacrificed at 28 d.p.i. exhibited characteristic clinical signs associated with MoMuSV-349, including moderate cachexia. Histologically, neoplasms observed at 28 d.p.i. contained a significant vascular component. By 35 d.p.i., all mice exhibited severe clinical signs (e.g. cachexia, dull hair coat, uneven gait). Histologically, all the neoplasms had a predominant vascular component. Non-neoplastic lesions, such as severe thymic atrophy and multifocal pulmonary haemorrhage, were commonly present. Mice sacrificed 42 d.p.i. were clinically, grossly and histologically similar to those sacrificed at 35 d.p.i. However, one difference found in the 42 d.p.i. group was the presence of rare rhabdomyosarcomas infiltrating the skeletal muscles. Mice inoculated with MoMuSV-349 developed severe neutrophilia and lymphopenia, and moderate anaemia. This study demonstrates that MoMuSV-349 induced angiosarcomatous neoplasms are characterized by stage development and severe haematologic and non-neoplastic abnormalities.

The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified.

Tumor cells expressing the herpes simplex virus thymidine kinase (HSV-TK) gene are sensitive to the drug ganciclovir (GCV). We demonstrate here that HSV-TK-positive cells exposed to GCV were lethal to HSV-TK-negative cells as a result of a "bystander effect." HSV-TK-negative cells were killed in vitro when the population of cultured cells contained only 10% HSV-TK-positive cells. The mechanism of this "bystander effect" on HSV-TK-negative cells appeared to be related to the process of apoptotic cell death when HSV-TK-positive cells were exposed to GCV. Flow cytometric and electron microscopic analyses suggested that apoptotic vesicles generated from the dying gene-modified cells were phagocytized by nearby, unmodified tumor cells. Prevention of apoptotic vesicle transfer prevented the bystander effect. The toxic effect of HSV-TK-positive cells on HSV-TK-negative cells was reproduced in an in vivo model. A mixed population of tumor cells consisting of HSV-TK-positive and HSV-TK-negative cells was inoculated s.c. into mice. Regression of the tumor mass occurred when the inoculum consisted of as few as 10% HSV-TK-expressing tumor cells. The bystander effect was also demonstrated in i.p. tumor studies. Initial experiments demonstrated that prolonged survival (> 70 days) occurred when a mixture containing 50% HSV-TK-positive and 50% HSV-TK-negative cells was injected i.p. followed by GCV treatment. Further, survival was prolonged for mice with a preexisting HSV-TK-negative i.p. tumor burden by injecting HSV-TK-positive cells and GCV. These results suggest that genetic modification of tumor cells may be useful for developing cancer therapies.

Ultrastructural localization of lectin-binding sites in different basement membranes.

In the present work we localized binding sites for the lectins WGA, RCA I, con A and SBA at the ultrastructural levels in morphologically different basement membranes. These different basement membranes included (a) thin ones, for example, tubular basement membrane of the mouse kidney which separates epithelial cell layers from mesenchymal cells and glomerular basement membrane which separates epithelial cells from other epithelial cells, (b) thick multilayered ones, for example. Reichert's membrane which is built up during the embryonic development of rodents and as an example of a pathologically thickened basement membrane, the basement membrane of the Engelbreth-Holm-Swarm (EHS) sarcoma. We were able to show that, in contrast to the thick multilayered basement membranes, the thin ones showed a strong positive SBA-binding pattern. Thick basement membranes otherwise revealed very strong labelling with the lectins WGA and RCA I. Our findings lead us to conclude that thin and thick basement membranes differ markedly in the quality and quantity of the carbohydrates which they contain.