Christine Helige

Uterine Natural Killer Cells in a Three-Dimensional Tissue Culture Model to Study Trophoblast Invasion

The high numbers of CD56+ cells with natural killer (NK) functions present in the uterine mucosa during the late secretory phase of the menstrual cycle and during early pregnancy have been considered to be implicated in implantation and in the regulation of trophoblast invasion. A three-dimensional organ culture model was used to study the interactions of these uterine NK cells with Jeg-3 and BeWo choriocarcinoma cells as a model of the invasive trophoblast. For this purpose, fragments of endometrial and decidual tissue were put in close contact with multicellular spheroids of choriocarcinoma cells in small silicon funnels. After the formation of stable contacts, the confrontation cultures were transferred to spinner flasks, cultivated for up to 6 days, and prepared for immunohistochemistry. During 2 days of cocultivation, the first cells started to move forward into the stromal component of the confrontation culture as demonstrated by staining of the choriocarcinoma cells using anti-human cytokeratin. Invasion advanced until, after a total of 6 days, some choriocarcinoma cells had already penetrated deeply into the host tissue. After a cultivation period of 1 week, both the endometrial and decidual tissue fragments still contained several CD56+ uterine NK cells, and some of them expressed the proliferation-associated marker Ki-67 without any exogenous activation. A few CD56+ cells were found directly at the invasion front, as well as between the choriocarcinoma cells. These cells also contained the cytolytic granule protein perforin indicating a migration of NK cells with cytolytic potential toward the potentially invasive cells. In conclusion, this human system closely resembles the in vivo conditions during trophoblast invasion and provides an appropriate in vitro model for studying dynamic processes involving various cell types during trophoblast invasion at the experimental level. Moreover, it enables us to study the effects of cytokines and growth factors that possibly regulate trophoblast invasion.

Effect of dequalinium on K1735-M2 melanoma cell growth, directional migration and invasion in vitro.

Cationic lipophilic compounds have an antiproliferative effect on certain tumour systems in vitro and in vivo. We have investigated whether the cationic lipophilic compound dequalinium affects not only proliferation but also motility and invasion of the highly metastatic and highly invasive melanoma cell line K1735-M2. Proliferation was assessed in monolayer cultures and in multicellular spheroids, motility was estimated in the assay of directional migration, and invasiveness was tested through confrontation cultures of tumour multicellular spheroids with embryonic chick heart tissue evaluated by computerized image analysis. 2 mumol/l dequalinium impaired melanoma cell proliferation, reduced directional migration and significantly blocked invasion in vitro. On the ultrastructural level, dequalinium caused obvious changes in mitochondria of both melanoma and embryonic chick heart cells. The mechanisms of the antiproliferative, antimigrating and antiinvasive effects remain to be determined. Inhibition of protein kinase C, calmodulin antagonism, DNA intercalation and/or direct effects on mitochondrial functions may be considered.

Correlation of melanoma cell motility and invasion in vitro.

Cell motility and the ability to grow invasively are crucial properties within the metastatic cascade. The relation of cell motility in vitro and metastatic behaviour of tumour cells in animal experiments indicates that they are directly correlated. We undertook this study to see whether a quantitative correlation could be found in complex in vitro systems. Using the assay of directional migration and a newly developed image analysis system to measure cell motility of K1735-M2 mouse melanoma cells and the embryonic chick heart assay of Mareel to follow invasion, we examined the influence of eight compounds on cell motility seven compounds on invasion. For stationary motility we calculated the change of density, area of change, area of ruffling sites (representing only changes at the leading edge and tail of the cell), number of ruffling sites, area of changing intracellular particles and number of intracellular particles. Velocity of single tumour cells and directional migration were also measured. In the invasion assay the parameters STRCSTR and INVASLOG, expressing different forms of stromal (i.e. embryonic chick heart) disintegration and degradation, were calculated. Directional migration and all parameters of stationary motility except number of ruffling sites, changing intracellular particles and number of changing intracellular particles correlated significantly (p<0.05) with STRCSTR and INVASLOG. For velocity, area of change and area of ruffling we found the most significant correlation with parameters of invasion indicating that both stationary and translocative motility contribute to invasion. Our systems also showed that the compounds tested exerted differential effects on various aspects of motility. Thus, there appears to be a strong correlation between cell motility and invasive growth in vitro Discrimination of the different kinds of stationary motility by image analysis allows detailed analysis of the influence of different compounds on cell motility of melanoma cells in vitro Quantification of single cell motility, radial migration and invasion can be useful in the evaluation of pharmacologic effects of new antimetastatic drugs interacting with cell motility and tumour cell invasion.

Inhibition of melanoma cell directional migration in vitro via different cellular targets

Abstract In malignant melanoma active movement of cancer cells is considered to be essential for tissue invasion. Various mechanisms, such as the Ca2+‐calmodulin‐proteinkinase C cascade or G‐protein‐dependent processes are considered to play a role in tumor cell functions. The assay of directional migration, combined with computer‐assisted image analysis, was used to evaluate the antimigratory efficacy of drugs interfering with different steps of signal (ransduction pathways. Treatment with different compounds showed a more or less concentration‐dependent reduction of migration rates: The Ca2+‐channel blockers verapamil and devapamil showed a slight reduction of molility. The effect was more pronounced when the calmodulin antagonist flunarizine was used or the proteinkinase C inhibitors dequalinium, tamoxifen and H‐7 were applied. A marked inhibition of molility was found with the G‐protein antagonist L 651582. Thus, our results indicate that different signal transduclion pathways are involved in the regulation of directional migration of K1735‐M2 melanoma cells.

Quantitative assessment of melanoma single-cell motility in vitro

Abstract Cell motility is a crucial property of tumor cells during invasion and metastasis. In this study we developed a computer assisted system to measure translocation and stationary motility of single cells and used this procedure to evaluate the influence of cytochalasin A (CA) on single‐cell motility parameters of K1735‐M2 mouse melanoma cells. The cells were seeded at low density into a microincubator. Time lapse microcine‐malography was performed every 20 seconds from a high power field to assess stationary motility and every 10 minutes with a screening objective to measure translocalion. 1 μMol CA was added to the medium 48 hours before measurement. Calculation of stationary motility was performed by subtraction of subsequent images and the resulting image difference was used for quantitative evaluation. Three different measuring windows were drawn to discriminate between membrane ruffling, intra‐cellular organelle transport and overall stationary motility. For each cell we measured change of density (CD), area of change (AC), perimeter of area of change (PC), area of ruffling (AR), number of ruffling sites (NR), change of inlracellular organelles (CIO) and number of changing intraccllular organelles (NIO). In order to quantify translocation, the center of gravity of each cell was assessed subsequently and the velocity was calculated by connecting the centers of gravity. CA‐treated cells showed a significantly lower stationary motility and membrane ruffling compared to the untreated cells (U‐test: p ≤ 0.01). but there was not significant difference concerning the intracellular organelle transport. The velocity of cells grown in the presence of CA (11.56, ±14.68 μm/h) was significantly lower (U‐test: p< = 0.01) than that of the control group (37.96, ±14.6 μm/h). In conclusion, our study shows that the techniques applied yield quantitative assessment of different characteristic parameters of single‐cell motility in vitro. Cytochalasin A significantly inhibited both membrane ruffling and translocative single‐cell motility of melanoma cells without impairing intracellular organelle transport.

Measurement of cell death by oxidative stress in three-dimensional spheroids from trophoblast and in fragments of decidua tissue

We report a new morphometric method for measurement of the amount of cell death in three-dimensional multicellular spheroids of the trophoblast-like cell line AC1-M59 and of cultured pieces of decidua tissue (decidua spheroids) in response to a cytotoxic agent. The viability of the spheroids was assessed by adding propidium iodide to the culture medium at the end of the toxic treatment. On fluorescence and brightfield images of serial cryosections the areas of propidium iodide fluorescence and the entire corresponding spheroids were measured by applying digital image processing and ratiometrical quantification. As an example, we evaluated the cytotoxic effect of hydrogen peroxide on both types of spheroids. The relative potency of hydrogen peroxide to induce tissue damage was assessed quantitatively for determination of the minimal concentration that leads to an increase in cytotoxicity. The method presented suggests general applicability for in vitro determination of toxicity against tissues.

Cytoplasmic microtubules in two different mouse melanoma cell lines: a qualitative and quantitative analysis using confocal laser scanning microscopy and computer‐assisted image analysis

The microtubular system as one part of the cellular cytoskeleton is not only necessary for mitotic activity of malignant cells but also for invading neighboring tissues and for the formation of distant metastases. In the present study, the amount and distribution of tubulin in two murine melanoma cell lines (K1735‐M2: high metastatic clone; K1735‐cl16: low metastatic clone) were determined quantitatively using an indirect immunofluorescence technique, confocal laser scanning microscopy (CLSM)and computer‐assisted image analysis. Additionally, qualitative and quantitative changes after application of the microtubule‐inhibitor nocodazole were investigated. Quantitative analysis showed a significant difference between the high and low metastatic cell line for the parameter TEXTURE, indicating a finer structured network within the high metastatic cells. After treatment with nocodazole the parameters TEXTURE and DENSITY were reduced, suggesting a decrease of assembled tubulin and a less delicate structure of the remaining microtubules. Our study shows that CLSM combined with computer‐assisted image analysis provides a new method to examine quantitative variations of the cytoskeleton possibly related to cell function.

Differential effects of synthetic sphingosine derivatives on melanoma cell motility, growth, adhesion and invasion in vitro

Cancer cell surface glycosphingolipids are considered to play a critical role in tumor growth and metastasis. However, the implications of glycoconjugates in the control of cell motility, which is considered to be involved in tumor invasion, are not fully understood. In this study, the effects of a series of synthetic sphingosine derivatives, obtained by the chemical transformation of azidosphingosines, on directional migration of K1735-M2 melanoma cells grown on type I collagen-coated surfaces were investigated. Following the application of 60 µm (2R, 3S, 4E)-2,3-epimino-4-octadecen-3-ol (S4) the migration rate was 94 ± 10 μm/day, compared with 377 ± 22 µm/day in the control experiment. Six other analogues were not as potent. S4 also considerably down-modulated melanoma single cell motility. Inhibition of motile activity was associated with changes in the actin filament organization as well as with changes in the number and distribution of vinculin plaques. Moreover, the compound reduced the attachment abilities of melanoma cells to basement membrane Matrigel. Tumor cell invasion, however, was less affected and proliferation remained unimpaired after treatment with S4. These data suggest at least one intracellular mode of action of this particular synthetic sphingosine derivative by modulation of cytoskeletal organization. Melanoma cell motility and growth may be controlled independently via glycosphingolipids.

Mofarotene-induced inhibition of melanoma cell motility by increasing vinculin-containing focal contacts.

Tumour cell motility, which is dependent on the organization of the cytoskeleton, is considered to play an important role in the spread of malignant melanoma. Therefore, retinoids, which are modulators of cytoskeletal organization, may affect the motile activity of melanoma cells. In this study, the effects of the arotinoid mofarotene on single cell motility and vinculin organization of the highly metastatic melanoma cell line K-1735-M2 were determined. Melanoma cells were cultivated in a temperature- and CO2-controlled microincubator, which was located on the microscope stage. Cell movements were evaluated quantitatively from time-lapse video recordings using an IBAS image analysis system. Vinculin distribution was evaluated using confocal laser scanning microscopy and a specially developed computerized image analysing program. In addition, melanoma cell invasion was tested on the embryonic chick heart model. Although 10 μM mofarotene did not reduce the translocative movements of melanoma cells, it significantly inhibited stationary motility, including fast plasma membrane movements and changes in shape. Mofarotene also showed a pronounced effect on the organization of vinculin-containing cell–substratum adhesion plaques. In retinoid-treated cells, the numbers of vinculin plaques per cell, and particularly those in the marginal areas of the cells, were significantly increased compared with untreated controls. Furthermore, the compound reduced the invasiveness of melanoma cells in a three-dimensional tissue culture model. In conclusion, our data demonstrate that mofarotene, an already almost forgotten synthetic retinoid, shows interesting effects on melanoma cells, which may be relevant for a slowdown of tumour spread.