DNA ploidy in curettage specimens identifies high-risk patients and lymph node metastasis in endometrial cancer.

BACKGROUND: Preoperative risk stratification is essential in tailoring endometrial cancer treatment, and biomarkers predicting lymph node metastasis and aggressive disease are aspired in clinical practice. DNA ploidy assessment in hysterectomy specimens is a well-established prognostic marker. DNA ploidy assessment in preoperative curettage specimens is less studied, and in particular in relation to the occurrence of lymph node metastasis. METHODS: Curettage image cytometry DNA ploidy in relation to established clinicopathological variables and outcome was investigated in 785 endometrial carcinoma patients prospectively included in the MoMaTEC multicentre trial. RESULTS: Diploid curettage status was found in 72.0%, whereas 28.0% were non-diploid. Non-diploid status significantly correlated with traditional aggressive postoperative clinicopathological features, and was an independent predictor of lymph node metastasis among FIGO stage I-III patients in multivariate analysis (OR 1.94, P=0.033). Non-diploid status was related to shorter disease-specific survival (5-year DSS of 74.4% vs 88.8% for diploid curettage, P<0.001). When stratifying by FIGO stage and lymph node status, the prognostic effect remained. However, in multivariate regression analysis, preoperative histological risk classification was a stronger predictor of DSS than DNA ploidy. CONCLUSIONS: Non-diploid curettage is significantly associated with aggressive clinicopathological phenotype, lymph node metastasis, and poor survival in endometrial cancer. The prognostic effect was also observed among subgroups with (presumably) less aggressive traits, such as low FIGO stage and negative lymph node status. Our results indicate curettage DNA ploidy as a possible supplement to existing parameters used to tailor surgical treatment.

Hypoxia-inducible factor-1alpha is an intrinsic marker for hypoxia in cervical cancer xenografts.

The hypoxia-inducible factor 1 (HIF-1) is known to induce the expression of several proteins linked to the maintenance of oxygen homeostasis, cellular energy metabolism, and tumor progression. Its alpha subunit (HIF-1alpha) is stabilized under hypoxic conditions and, therefore, might represent an intrinsic marker for tissue hypoxia. Here we report on the spatial relationship between HIF-1alpha and the nitroimidazole hypoxia marker EF5 in cervical carcinoma xenografts, and on their spatial relationship to tumor blood vessels. EF5 was administered to mice bearing ME180 and SiHa cervical cancer xenografts. Frozen tumor tissue sections, triple-stained for HIF-1alpha, the endothelial cell marker CD31, and EF5, were imaged using wide-field multiparameter immunofluorescence microscopy. Expression levels of EF5 and HIF-1alpha were similar in ME180 xenografts, but the percentage of tumor area stained with EF5 was significantly smaller than the percentage of HIF-1alpha-positive area in SiHa tumors. In both tumor types the EF5-HIF-1alpha overlap was statistically significant, thus confirming their spatial and temporal colocalization. Spatial distribution analysis of EF5 and HIF-1alpha is consistent with different pO2 value "thresholds" for EF5 binding and HIF-1alpha expression. Summarized, our results indicate that HIF-1alpha is a useful intrinsic marker for hypoxia in cervical carcinoma xenografts.

Preservation of the ependyma of the mouse spinal cord by various vascular perfusion procedures.

Seventy-two mice were used to find out which of 13 vascular perfusion procedures gave best structural preservation of the spinal cord ependyma and central canal lumen. Best results were obtained by 3% glutaraldehyde in Tyrodes solution with 50% of normal NaCl amount, 0.06 M sucrose and 2% dextran T-40 (556 mOsmol, pH 7.2, 0-4 degrees C). This was perfused by a peristaltic pump at 40 ml/min for 10 min through a cannula inserted in the ascending aorta. No advantage was seen by heparin pretreatment or adding a prefixation rinse. With good tissue preservation the central canal was found to be round to oval in cross-sectional profile and almost free of intraluminal material.

Laminin expression by glial fibrillary acidic protein positive cells in human gliomas.

Extracellular matrix components are regarded as important substrates for invasive tumor cells. The present work focuses on the expression of laminin in the brain in response to invading brain tumors. Biopsies obtained from tissue macroscopically evaluated as the border zone between tumor and normal brain, in 5 patients undergoing surgery for glioblastoma multiforme, were examined by immunocytochemistry and scanning confocal microscopy for the expression of laminin and glial fibrillary acidic protein. Laminin was mainly found in all the specimens associated with the basal lamina of blood vessels, but a variable degree of punctate laminin deposits were also observed in the parenchyma not associated with blood vessels. In the specimens with substantial deposits, scanning confocal microscopy showed that some of the laminin co-localized with intracellular glial fibrillary acidic protein. Punctate deposits of laminin were also seen in an intracranial BT4C rat glioma model, where it was particularly abundant in the brain/tumor confrontation zone. Previous in vitro studies have shown that laminin, among several extracellular matrix components, represent a highly permissive substrate for glioma cell migration. The presented results indicate that laminin can be produced by glial fibrillary acidic protein positive cells during glioma cell invasion in humans. This glycoprotein may thus represent one important substrate among many, which contribute to the invasive phenotype of gliomas.

Pharmacological manipulation with the descending serotonergic system or transection of the mouse spinal cord has no effect on ependymal ultrastructure.

In order to investigate an effect of descending nerve fibres on mouse spinal cord ependymal ultrastructure, pharmacological manipulation with the serotonergic system or transection of the spinal cord was done. Biochemical analysis showed an 83% reduction of serotonin content in spinal cord tissue after p-chlorophenylalanine injections and a 93% reduction after transection. However, none of the experimental animals showed changes in ependymal ultrastructure compared to control animals as revealed by electron microscopy.

Imaging of whole tumor cut sections using a novel scanning beam confocal fluorescence MACROscope.

Hypoxia caused by inadequate structure and function of the tumor vasculature has been found to negatively determine the prognosis of cancer patients. Hence, understanding the biological basis of tumor hypoxia is of significant clinical interest. To study solid tumor microenvironments in sufficient detail, large areas (several mm in diameter) need to be imaged at microm resolutions. We have used a novel confocal scanning laser MACROscope (CSLM) capable of acquiring images over fields of view up to 2cm x 2cm. To demonstrate its performance, frozen sections from a cervical carcinoma xenograft were triple labeled for tissue hypoxia, blood vessels and hypoxia-inducible transcription factor 1 alpha (HIF-1alpha), imaged using the CSLM and compared to images obtained using a standard epifluorescence microscope imaging system. The results indicate that the CSLM is a useful instrument for imaging tissue-based fluorescence at resolutions comparable to standard low-power microscope objectives.

Differential effects of vincristine and phenytoin on the proliferation, migration, and invasion of human glioma cell lines.

The aim of this study was to investigate the antimigratory and antiinvasive potential of vincristine sulfate (VCR) on human glioma cells and to analyze whether phenytoin (5,5-diphenylhydantoin; DPH) might act synergistically with VCR. Vincristine affects the cytoplasmic microtubules; DPH has been reported to enhance VCR cytotoxicity in murine cells. In two human glioma cell lines, GaMG and D-37MG, we found VCR to reduce monolayer growth and colony formation in a dose-dependent fashion at concentrations of 10 ng/ml and above. Phenytoin increased the cytotoxic and cytostatic effects of VCR in monolayer cells but not in spheroids. Multicellular spheroids were used to investigate directional migration. A coculture system of GaMG and D-37MG spheroids with fetal rat brain aggregates was used to analyze and quantify tumor cell invasion. A dose-dependent inhibition of migration and invasion by VCR was observed in both cell lines without further enhancement by DPH. Immunofluorescence microscopy with antibodies against alpha-tubulin revealed dose-dependent morphological alterations in the microtubules when the cells were exposed to VCR but not after incubation with DPH. Based on the combination of standardized in vitro model systems currently in use and the present data, the authors strongly suggest that VCR inhibits migration and invasion of human glioma cells. This is not altered by DPH, which inhibits cell proliferation in combination with VCR.

Dynamic determination of human glioma invasion in vitro.

OBJECT: The goal of this study was to evaluate whether there is any relationship between survival of patients with brain tumor and tumor proliferation or tumor invasion in vitro. METHODS: Samples of freshly resected brain tumors from 14 patients with glioblastoma multiforme (GBM) were directly grown as three-dimensional multicellular spheroids. The tumor spheroids were cocultured with fetal rat brain cell aggregates (BCAs), used to represent an organotypical normal brain tissue model. Before the coculture, the tumor spheroids and the BCAs were stained with two different carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 3,3'-dioctadecycloxacarbocyanine perchlorate (DiO), respectively. During the coculture, confocal laser scanning microscopy allowed a sequential analysis of tumor cell invasion by visualizing dynamic aspects of the invasive process. Single cocultures were examined at three different time points (24, 48, and 96 hours). During the observation period there was a change in the structural morphology of the cocultures, with a progressive decrease in BCA volume. Furthermore, the scanning confocal micrographs revealed a bidirectional movement of tumor cells and normal cells into brain and tumor tissue, respectively. It is also shown that there is a considerable variation in the rate of BCA destruction in cocultures of glioma spheroids generated directly from biopsy specimens. This variation is seen both between spheroids generated from the same biopsy as well as between spheroids that are grown from different biopsy specimens. Cell proliferation measured by Ki-67 immunohistochemical analysis of biopsy samples obtained in the same patients revealed a correlation between tumor cell proliferation and tissue destruction of the BCAs, as determined by a reduction in BCA volume (p = 0.0338). No correlation was found when survival was related to the same parameters (p > 0.05). CONCLUSIONS: The present work provides a model for quick and efficient assessment of dynamic interactions between tumor and normal brain tissue shortly after surgery.

Combined effect of alkyl-lysophospholipid and vincristine on proliferation, migration and invasion in human glioma cell lines in vitro.

The aim of the present study was to investigate the combined effect of the alkyl-lysophospholipid Et-18-0-CH3 (ALP) and vincristine (VCR) on the proliferative, migratory and invasive properties of human glioma cells. Both drugs have previously been shown to act upon the locomotive apparatus of the cell. In the glioma cell lines D37MG and GaMG we found that ALP and VCR inhibited proliferation in monolayer growth as well as in multicellular spheroids in a dose-dependent manner. When combined, the two drugs had a significantly enhanced effect upon proliferation in the cell line GaMG but not in D37MG. Migration was inhibited by ALP and VCR in a dose-dependent manner in both cell lines. An enhanced effect by combining the drugs was also observed. The invasion of glioma cells into the brain cell aggregates was markedly inhibited at lower concentrations than needed for the inhibition of proliferation and migration when ALP and VCR were applied alone, without any further enhancement by combining the drugs. It is concluded that the combination of ALP and VCR have additive anti-proliferative and anti-migratory properties in human glioma cell lines.

Adhesion and migration of human glioma cells are differently dependent on extracellular matrix molecules.

To analyse the interactions between glioma cells and extracellular matrix (ECM) proteins, the adhesive and migratory capacity of five human glioma cell lines (D37MG, D54MG, GaMG, U118MG and U251MG) were studied. The expression of integrins was analysed and correlated to the adhesive and migratory abilities of the cells. All cell lines were able to adhere to and migrate on the extracellular matrix proteins collagen IV, fibronectin, laminin and vitronectin. Laminin was superior in propagating adhesion and migration in all five cell lines. As analysed by flow cytometry, the expression of the integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, beta 3, beta 4, alpha v, and integrin alpha v beta 5 proved to be uniform between the cell lines. All integrins except alpha 4, alpha 6, beta 3 and beta 4 were expressed on more than 85% of the cells. Inhibition of adhesion with synthetic peptides and antibodies directed against integrins demonstrated that adhesion on laminin was independent of integrins and the 67 kD laminin receptor. On the other hand, migration was shown to be integrin-dependent on all substrates. The results indicate that the mechanism responsible for cell adhesion in human gliomas differ from those present during migration, and that integrins play an important role in regulating these two mechanisms.

ECM dependent and integrin mediated tumor cell migration of human glioma and melanoma cell lines under serum-free conditions.

Collagen IV, laminin and fibronectin are constituents of the cerebral extracellular matrix (ECM), which is critical in glioma cell invasion. The aim of the present study was to evaluate the integrin dependent cell-matrix interactions of two tumors with different invasive properties under matrixfree conditions. Two human glioma (GaMG, U373) and melanoma (MV3, BLM) cell lines were grown in serum free medium. Immunofluorescence microscopy of collagen IV, laminin, and fibronectin was performed. The adhesion of monolayer cells and their migration out of multicellular spheroids was quantified for these ECM components. Integrin chains known to act as laminin receptors were blocked by specific antibodies in additional migration assays. All cell lines expressed all the ECM components under serum free conditions. Tumor cell adhesion and migration in both glioma and melanoma cell lines was increased by all the ECM components, laminin being the strongest promotor of migration. However, migration was dose dependent in gliomas, whereas melanomas revealed a dose optimum of 10 micrograms/ml laminin. Antibodies against alpha 3 integrins significantly reduced migration on laminin in all cell lines, anti-beta 1 in all cell lines except U373. Anti-alpha 2 in BLM showed a strong effect, anti-alpha 6 was a stronger inhibitor in glioma than in melanoma cells. Integrins are functionally involved in tumor cell locomotion on laminin. The blocking of laminin related integrin chains markedly reduces cell motility in a varying manner between the cell lines. Moreover, different cell lines utilize different integrins as the laminin receptor.

Heterogeneic modulation of malignant behavior in human glioma cells in defined and serum-containing media.

Malignant features in three glioma cell lines were studied in four defined media of various complexity. The cell lines D37MG, D54MG, and GaMG were able to grow in monolayer culture in all media examined, and as multicellular tumor spheroids in the two most nutrient-rich media. In the defined media, none of the cell lines were able to migrate in a migration assay on poly-D-lysine-coated plastic surfaces. Flow cytometric analysis of the GaMG cell line demonstrated no medium-dependent selection of subclones of glioma cells in spheroids cultured for 30 d. Morphological diversity of spheroids varied according to the supplementation of the media. The capacity of glioma cells to invade cellular rat brain aggregates was intact in the media examined. However, glioma migration was severely inhibited by the lack of specific serum components. This study demonstrates that glioma growth and invasion was heterogeneously preserved in the defined media used. Depending on the assay to be used in the study of glioma cell behavior, the degree of medium supplementation has to be considered.

Epidermal growth factor and laminin receptors contribute to migratory and invasive properties of gliomas.

Gliomas are characterized by their extensive invasion into the brain parenchyma. Recently it has been shown that normal brain cells can produce laminin, fibronectin and collagen type IV when confronted by invading glioma cells. Laminin stimulates cell migration of several human glioma cell lines in vitro. This migration can be inhibited by adding blocking monoclonal antibodies (MAbs) against the most expressed integrin subunits, alpha3 and beta1. Previous studies have shown that glioma cell migration, invasion and growth are stimulated by epidermal growth factor (EGF). However, MAb directed against the EGF receptor (EGFR) did only partly inhibit the invasive process in vitro. Since laminin has regional peptide homology with EGF (EGF-like repeats), the present work was aimed at studying how two human glioma cell lines exposed to antibodies to the EGFR, reacted to laminin stimulated migration. Furthermore, we wanted to study which role the EGFR and the laminin receptor integrin subunits alpha3 and beta1 play during glioma cell invasion. EGFR expression of two glioma cell lines, AN1/lacZ and U-251/lacZ was studied by flow cytometry and immunofluorescence microscopy. A cell migration assay was used to study effects of MAbs against EGFR on migration from laminin-stimulated tumor spheroids. Tumor cell invasion was evaluated by using an in vitro co-culture model, where normal fetal brain cell aggregates were confronted with multicellular tumor spheroids. The results show that both cell lines expressed EGFR, AN1/lacZ 4-fold more than U-251/lacZ. MAb against EGFR inhibited the laminin-stimulated migration only from AN1/lacZ spheroids. MAbs against alpha3 and beta1 integrin subunits inhibited glioma cell invasion in vitro. The present work indicates possible connections between laminin-stimulated cell migration and the EGFR expression on glioma cells. These elements contribute to the characteristic features of glioma cells and may be an important part of the complex relationships between growth factors, integrins and extracellular matrix during glioma cell invasion.

Ultrastructure of the mouse spinal cord ependyma.

This study was done in order to investigate the normal ultrastructure of well-preserved mouse spinal canal ependyma using light, scanning and transmission electron microscopy. The ependymal lining was found to consist of a simple, cuboidal epithelium essentially similar to the unspecialized cuboidal ependyma of the brain ventricles. Apart from great variation in kinociliary density, no intracellular difference was noted between the ependymal cells. In contrast to earlier findings, indications of the existence of zonulae occludentes between the apical part of the ependymal cells were observed. Our findings do not support the hypothesis of secretion or intracellular transport by the ependyma, or that the ependyma constitutes a significant diffusion barrier.

A stereological study of the ependyma of the mouse spinal cord. With a comparative note on the choroid plexus ependyma.

Applying different stereological techniques, the total ependymal volume in the spinal cord of mice was estimated to be 83 x 10(6) microns cubed, the number of cells to be 163,000 and the mean ependymal cell volume to be 510 microns cubed. Compared to choroid plexus cells in the third ventricle, the ependymal cells in the spinal cord contained a smaller mitochondrial volume (9.8% versus 4.6% of cell volume) and less rough endoplasmic reticulum (2.1% versus 0.4%). These findings indicate that the metabolic activity of the ependyma in the spinal cord is lower than that in the choroid plexus. Compared to liver and exocrine pancreatic cells, ependymal cells in both locations must be considered to have a rather low metabolic activity.

Expression of annexin II in glioma cell lines and in brain tumor biopsies.

Annexin II is a calcium and phospholipid binding protein and a substrate for protein-tyrosine kinases. Increased levels of annexin II are observed in various cancer cells and tissues, and the molecule has been proposed as a marker of malignancy in vivo. Annexin II was expressed in four glioma cell lines (D-54MG, D-37MG, U251MG and GaMG), as determined by Western blot analyses, immunofluorescence staining and flow cytometric measurements. In addition, annexin II expression was also found in cryostat sections obtained from 15 consecutive brain tumor biopsies: Ten were histologically classified as glioblastomas, one as an astrocytoma, two as meningiomas and two as brain metastases. Cultured spheroids from the glioma cell lines and from three of the glioblastoma biopsies showed lower levels of annexin II, than found in the monolayers of the cell lines and in the freshly cut biopsies. The annexin II expression of the cell lines were not found to be related to their proliferative, migratory or invasive properties. These findings indicate that although annexin II may serve as a marker of malignancy in vivo, its expression can be reduced in vitro, and appear unrelated to malignant features of glioma cell lines.