The in vitro influences of neurotensin on the motility characteristics of human U373 glioblastoma cells.

Astrocytic tumours are associated with dismal prognoses due to their pronounced ability to diffusely invade the brain parenchyma. Various neuropeptides, including gastrin, are able to modulate tumour astrocyte migration. While neurotensin has been shown to influence the proliferation of glioma cells and the migratory ability of a large set of other cell types, its role in glioma cell migration has never been investigated. Neurotensin-induced modifications to the motility features of human U373 glioblastoma cells therefore constitute the topic of the present study. We evidenced that three subtypes of neurotensin receptors (NTR1, NTR2 and NTR3) are expressed in U373 glioblastoma cells, at least as far as their mRNAs are concerned. Treating U373 tumour cells with 10 nM neurotensin markedly modified the morphological patterns of these cells and also profoundly altered the organization of their actin cytoskeletons. Pull-down assays revealed that neurotensin induced the activation in U373 cells of both Rac1 and Cdc42 but not RhoA. Scratch wound assays evidenced that neurotensin (0.1 and 10 nM) very significantly inhibited wound colonization by U373 cells cultured in the absence of serum. In addition, quantitative phase-contrast videomicroscopy analyses showed that neurotensin decreases the motility levels of U373 glioblastoma cells when these cells are cultured on plastic. In sharp contrast, neurotensin stimulates the motility of U373 cells when they are cultured on laminin, which is a pro-adhesive extracellular matrix component ubiquitously secreted by glioma cells. Our data thus strongly suggest that, in addition to gastrin, neurotensin is a neuropeptide capable of modulating tumour astrocyte migration into the brain parenchyma.

The infrared spectrum of human glioma cells is related to their in vitro and in vivo behavior.

The present research investigates whether infrared spectra can be related to the biological characteristics of glioma cell lines. We used nine human glioma cell lines for which a series of in vitro and in vivo biological features had already been established [Glia 36 (2001) 375] and were able to show that their characteristic infrared spectra reflect their in vitro migration (i.e., motility and invasiveness) properties and their in vivo aggressiveness. More particularly, the infrared data evidenced correlations at the level of the lipid/protein ratio. These relationships were found to be tissue-dependent when controlled on seven pancreatic carcinoma cell lines. We also showed that oligodendroglial and astrocytic tumor cells, whose identification remains difficult, can easily be identified by their infrared spectra in the lipid acyl chain region as well as in the nucleic acid region. We concluded that infrared spectroscopy could usefully complement information provided by more conventional diagnostic and prognostic (e.g., morphological and molecular) approaches.

Glycohistochemical characterization of vascular muscle cell destruction in CADASIL subjects by lectins, neoglycoconjugates and galectin-specific antibodies.

CADASIL (Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is a type of small-artery stroke and vascular dementia-inducing pathology of the brain. In order to explain the molecular mechanisms behind the alterations to the blood vessels in CADASIL subjects, we scrutinized the expression of glycan and glycan-binding sites in the wall of vessels taken from five such subjects (vs. five control subjects matched for age and sex). Specimens were taken from the brain, heart, kidney, liver and lung. Although the main vessel lesions were observed in the tissues depending on the blood-brain barrier, alterations to systemic vessels were also observed despite the absence of any symptoms. The histochemical expression of a panel of 10 biotinylated neoglycoconjugates [Gal-beta(1-4)-D-Glc, Galbeta(1-3)GalNAc, alpha-D-GalNAc, beta-D-GalNAc, GalNAcalpha(1-3)-D-GalNAcalpha, GalNAcalpha(1-3)-D-GalNAcbeta, beta-D-Glc, alpha-D-Man, l-Fucose and D-Glcalpha(1-4)-D-Glc], eight plant lectins (PNA, MAA, SNA, DBA, WGA, ConA, GNA and UEA-1) and two antigalectin antibodies was monitored by means of semiquantitative and quantitative computer-assisted microscopy. The data show the altered histochemical binding of plant lectins, such as UEA-1 and ConA, in the vessel walls of CADASIL subjects. The present work, based upon staining by a panel of neoglycoconjugates, provides a biochemical characterization of the alteration of vessel walls in the brain compared to other organs including the heart, kidney, lung and liver in CADASIL as opposed to control subjects. These glycohistochemical results suggest a functional relevance of protein-carbohydrate interactions in this disease.

Galectin-8 expression decreases in cancer compared with normal and dysplastic human colon tissue and acts significantly on human colon cancer cell migration as a suppressor.

BACKGROUND AND AIMS: Galectins are beta-galactoside binding proteins. This ability may have a bearing on cell adhesion and migration/proliferation in human colon cancer cells. In addition to galectins-1 and -3 studied to date, other members of this family not investigated in detail may contribute to modulation of tumour cell features. This evident gap has prompted us to extend galectin analysis beyond the two prototypes. The present study deals with the quantitative determination of immunohistochemical expression of galectin-8 in normal, benign, and malignant human colon tissue samples and in four human colon cancer models (HCT-15, LoVo, CoLo201, and DLD-1) maintained both in vitro as permanent cell lines and in vivo as nude mice xenografts. The role of galectin-8 (and its neutralising antibody) in cell migration was investigated in HCT-15, LoVo, CoLo201, and DLD-1 cell lines. METHODS: Immunohistochemical expression of galectin-8 and its overall ability to bind to sugar ligands (revealed glycohistochemically by means of biotinylated histochemically inert carrier bovine serum albumin with alpha- and beta-D-galactose, alpha-D-glucose, and lactose derivatives as ligands) were quantitatively determined using computer assisted microscopy. The presence of galectin-8 mRNA in the four human colon cancer cell lines was examined by reverse transcriptase-polymerase chain reaction. In vitro, cellular localisation of exogenously added galectin-8 in the culture media of these colon cancer cells was visualised by fluorescence microscopy. In vitro galectin-8 mediated effects (and the influence of its neutralising antibody) on migration levels of living HCT-15, LoVo, CoLo201, and DLD-1 cells were quantitatively determined by computer assisted phase contrast microscopy. RESULTS: A marked decrease in immunohistochemical expression of galectin-8 occurred with malignancy development in human colon tissue. Malignant colon tissue exhibited a significantly lower galectin-8 level than normal or benign tissue colon cancers; those with extensive invasion capacities (T3-4/N+/M+) harboured significantly less galectin-8 than colon cancers with localised invasion capacities (T1-2/N0/M0). The four experimental models (HCT-15, LoVo, CoLo201, and DLD-1) had more intense galectin-8 dependent staining in vitro than in vivo. Grafting the four experimental human colon cancer models onto nude mice enabled us to show that the immunohistochemical expression of galectin-8 was inversely related to tumour growth rate. In vitro, galectin-8 reduced the migration rate of only those human experimental models (HCT-15 and CoLo201) that exhibited the lowest growth rate in vivo. CONCLUSIONS: Expression of galectin-8 correlated with malignancy development, with suppressor activity, as shown by analysis of clinical samples and xenografts. In vitro, only the two models with low growth rates were sensitive to the inhibitory potential of this galectin. Future investigations in this field should involve fingerprinting of these newly detected galectins, transcending the common focus on galectins-1 and -3.

Molecular characterization of cell substratum attachments in human glial tumors relates to prognostic features.

Glioma cell attachments to substratum play crucial roles in the invasion by glioma cells of normal brain tissue. These attachments are mediated through interactions between extracellular matrix (ECM) components, integrins, focal adhesion-linked molecules, and the actin cytoskeleton. In the present study, we investigate the molecular elements involved in cell substratum attachments in human glial tumors and their potential relationships to prognostic features. We used 10 human glioma cell lines, for which we characterized glial differentiation by means of quantitative RT-PCR for nestin, vimentin, and GFAP mRNA. We quantitatively determined the amounts of laminin, fibronectin, vitronectin, and thrombospondin secreted by these glioma cell lines in vitro, as well as the amount of each of the eight beta integrin subunits and the adhesion complex-related molecules, including talin, vinculin, profilin, zyxin, alpha-actinin, paxillin, and VASP. After quantification of the levels of migration and invasion of these 10 cell lines in vitro and, through grafts into the brains of nude mice, of their biological aggressiveness in vivo, it appeared that the levels of the beta 5 integrin subunit and alpha-actinin were directly related to biological aggressiveness. These experimental data were clinically confirmed because increasing immunohistochemical amounts of the beta 5 integrin subunit and alpha-actinin were directly related to dismal prognoses in the case of astrocytic tumors. In addition, we show that the beta 4 integrin subunit are expressed significantly more in oligodendrogliomas than in astrocytic tumors. A potential role for the beta 8 integrin subunit in glioma cell substratum attachments is also emphasized.

Gastrin induces over-expression of genes involved in human U373 glioblastoma cell migration.

Astrocytic tumors are the most common and the most malignant primary tumors of the central nervous system. We had previously observed that gastrin could significantly modulate both cell proliferation and migration of astrocytoma cells. We have investigated in the present study which genes could be targeted by gastrin in tumor astrocyte migration. Using a subtractive hybridization PCR technique we have cloned genes differentially over-expressed in human astrocytoma U373 cells treated or not with gastrin. We found about 70 genes over-expressed by gastrin. Among the genes overexpressed by gastrin, we paid particular attention to tenascin-C, S100A6 and MLCK genes because their direct involvement in cell migration features. Their gastrin-induced overexpression was quantitatively determined by competitive RT-PCR technique. We also showed by means of a reporter gene system that S100A6 and tenascin-C respective promoters were upregulated after gastrin treatment. These data show that gastrin-mediated effects in glioblastoma cells occur through activation of a number of genes involved in cell migration and suggest that gastrin could be a target in new therapeutic strategies against malignant gliomas.

S100A2, a putative tumor suppressor gene, regulates in vitro squamous cell carcinoma migration.

It has been previously shown that S100A2 is down-regulated in tumor cells and can be considered a tumor suppressor. We have recently shown that this down-regulation can be observed particularly in epithelial tissue, where S100A2 expression decreases remarkably in tumors as compared with normal specimens. In the present paper we investigate whether S100A2 could play a tumor-suppressor role in certain epithelial tissues by acting at the cell migration level. To this end, we made use of five in vitro human head and neck squamous cell carcinoma lines in which we characterized S100A2 expression at both RNA and protein level. To characterize the influence of S100A2 on cell kinetic and cell motility features, we used two complementary approaches involving specific antisense oligonucleotides and the addition of S100A2 to the culture media. The different expression analyses gave a coherent demonstration of the fact that the FADU and the RPMI-2650 cell lines exhibit high and low levels of S100A2 expression, respectively. Antisense oligonucleotides (in FADU) and extracellular treatments (in RPMI) showed that, for these two models, S100A2 had a clear inhibitory influence on cell motility while modifying the cell kinetic parameters only slightly. These effects seem to be related, at least in part, to a modification in the polymerization/depolymerization dynamics of the actin microfilamentary cytoskeleton. Furthermore, we found evidence of the presence of the receptor for advanced glycation end-products (RAGE) in RPMI cells, which may act as a receptor for extracellular S100A2. The present study therefore presents experimentally based evidence showing that S100A2 could play a tumor-suppressor role in certain epithelial tissues by restraining cell migration features, at least in the case of head and neck squamous cell carcinomas.

Galectin-1 is highly expressed in human gliomas with relevance for modulation of invasion of tumor astrocytes into the brain parenchyma.

Protein (lectin)-carbohydrate interaction is supposed to be relevant for tumor cell behavior. The aims of the present work are to investigate whether galectin-1 modulates migration/invasion features in human gliomas in vitro, whether it can be detected in human gliomas immunohistochemically, and whether its expression is attributable to certain glioma subgroups with respect to invasion and prognosis. For this purpose, we quantitatively determined (by computer-assisted microscopy) the immunohistochemical expression of galectin-1 in 220 gliomas, including 151 astrocytic, 38 oligodendroglial, and 31 ependymal tumors obtained from surgical resection. We also xenografted three human glioblastoma cell lines (the H4, U87, and U373 models) into the brains of nude mice in order to characterize the in vivo galectin-1 expression pattern in relation to tumor invasion of the normal brain parenchyma. In addition, we characterized the role in vitro of galectin-1 in U373 tumor astrocyte migration and kinetics. Our data reveal expression of galectin-1 in all human glioma types with no striking differences between astrocytic, oligodendroglial, and ependymal tumors. The level of galectin-1 expression correlated with the grade in the group of astrocytic tumors only. Furthermore, immunopositivity of high-grade astrocytic tumors from patients with short-term survival periods was stronger than that of tumors from patients with long-term survivals. In human glioblastoma xenografts, galectin-1 was preferentially expressed in the more invasive parts of these xenografts. In vitro experiments revealed that galectin-1 stimulates migration of U373 astrocytes.

Distinct differences in binding capacity to saccharide epitopes in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas, and glioblastomas.

We monitored the expression of glycan-binding sites on a panel of 10 biotinylated neoglycoconjugates by means of quantitative computer-assisted microscopy to further study the molecular mechanisms in the extensive infiltration of the surrounding brain parenchyma by most astrocytic tumors. Three distinct histological compartments were analyzed for each of the 108 astrocytic tumors (15 pilocytic astrocytomas (WHO grade I), 25 astrocytomas (WHO grade II), 30 anaplastic astrocytomas (WHO grade III), and 38 glioblastomas (WHO grade IV) included in our series. These compartments were tumors (nonperivascular tumor astrocytes), perivascular tumor astrocytes, and blood vessel walls. Clear differences were observed between the pilocytic and the diffuse astrocytic tumors. Furthermore, malignant progression in the latter category was paralleled by a decrease in cells' ability to bind distinct sugar epitopes, especially the D-GalNAc(alpha1-3)-D-GalNAc-beta1-R determinant of the Forssman pentasaccharide in tumors, the alpha-L-fucose in perivascular tumor areas, and the beta-D-glucose in tumor vessel walls. Markedly, the level of binding site expression for alpha-D-mannose decreased in the tumors, the perivascular tumor areas, and the vessel walls. These glycohistochemical results imply the functional relevance of protein-carbohydrate interactions in this tumor system.

Galectins are differentially expressed in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas, and significantly modulate tumor astrocyte migration.

Galectins, a family of mammalian lectins with specificity to beta-galactosides, are involved in growth-regulatory mechanisms and cell adhesion. A relationship is assumed to exist between the levels of expression of galectins and the level of malignancy in human gliomas. A comparative study of this aspect in the same series of clinical samples is required to prove this hypothesis. Using computer-assisted microscopy, we quantitatively characterized by immunohistochemistry the levels of expression of galectins-1, -3 and -8 in 116 human astrocytic tumors of grades I to IV. Extent of transcription of galectins-1, -3, and -8 genes was investigated in 8 human glioblastoma cell lines by means of RT-PCR techniques. Three of these cell lines were grafted into the brains of nude mice in order to characterize in vivo the galectins-1, -3 and -8 expression in relation to the patterns of the tumor invasion of the brain. The role of galectin-1, -3 and -8 in tumor astrocyte migration was quantitatively determined in vitro by means of computer-assisted phase-contrast videomicroscopy. The data indicate that the levels of galectin-1 and galectin-3 expression significantly change during the progression of malignancy in human astrocytic tumors, while that of galectin-8 remains unchanged. These three galectins are involved in tumor astrocyte invasion of the brain parenchyma since their levels of expression are higher in the invasive parts of xenografted glioblastomas than in their less invasive parts. Galectin-3, galectin-1, and to a lesser extent galectin-8, markedly stimulate glioblastoma cell migration in vitro. Since bands for the transcripts of human galectins-2, -4 and -9 were apparently less frequent and intense in the 8 human glioblastoma cell lines, this system provides an excellent model to assign defined roles to individual galectins and delineate overlapping and distinct functional aspects.

Differential expression of S100 calcium-binding proteins characterizes distinct clinical entities in both WHO grade II and III astrocytic tumours.

The computer-assisted microscopic analysis of Feulgen-stained nuclei enabled us to identify two subgroups of astrocytomas (WHO grade II) and two subgroups of anaplastic astrocytomas (WHO grade III) with significantly distinct clinical outcomes (Decaestecker et al. Brain Pathol 1998; 8: 29-38). The astrocytomas labelled in the present study as typical (TYP-ASTs) behaved clinically like real astrocytomas while atypical astrocytomas (ATYP-ASTs) behaved similarly to anaplastic astrocytomas. The anaplastic astrocytomas that we labelled as typical (TYP-ANAs) behaved clinically like anaplastic astrocytomas while atypical ones (ATYP-ANAs) behaved like glioblastomas. In the present study, we investigate whether some biological characteristics could be evidenced across these four groups of TYP- and ATYP-ASTs and TYP- and ATYP-ANAs. The data show that the levels of expression (immunohistochemically assayed and quantitatively determined by means of computer-assisted microscopy) of vimentin, the glial fibrillary acidic protein and the platelet-derived growth factor-alpha did not differ significantly across these four groups of astrocytic tumours. The level of cell proliferation (determined by means of both the anti-proliferating cell nuclear antigen and the anti-MIB-1 antibodies; P < 0.001 to P < 0.0001) differed very significantly between the astrocytomas and anaplastic astrocytomas, but not between the typical and atypical variants identified in each group. In sharp contrast, the levels of expression of the S100A3 and S100A5 proteins differed markedly in the solid tumour tissue in relation to the astrocytic tumour types and grades. In addition, while the levels of expression of S100A6 did not change in the astrocytic tumour tissue in relation to histopathological grade, the levels of expression of this S100 protein (but not those of S100A3 and S100A5) differed markedly in the blood vessel walls according to whether these vessels originated from low- or high-grade astrocytic tumours.

Galectin-3 and galectin-3-binding site expression in human adult astrocytic tumours and related angiogenesis.

Using computer-assisted microscopy, the present work aimed to quantitatively characterize the level of the histochemically detectable expression of galectin-3 and galectin-3-binding sites in sections of a series of 84 astrocytic tumours (including 22 grade II, 21 grade III and 41 grade IV specimens) and seven non-tumoural specimens used as controls. The presence of galectin-3 and reactive sites for this lectin were monitored by means of a specific polyclonal anti-galectin-3 antibody (aGal3) and biotinylated galectin-3 (Gal3), respectively. The pattern of expression of galectin-3-binding sites is compared to the pattern of expression of laminin (a potential galectin-3 ligand) revealed using a biotinylated anti-laminin antibody (aLam). Three variables quantitatively characterizing histochemical staining reactions were evaluated by means of computer-assisted microscopy for each of the 3 probes under study (aGal3, Gal3 and aLam). The labelling index (LI) is the percentage of tissue area specifically stained by a histochemical probe. The mean optical density (MOD) denotes staining intensity. The concentration heterogeneity (CH) feature expresses the concentrational spread of individual fields. The data obtained in the present study show that: (i) white matter of a non-tumoural brain expresses galectin-3 (and also galectin-3-binding sites); (ii) the level of galectin-3 expression significantly decreases in the majority of tumour astrocytes from low to high grade astrocytic tumours; while (iii) some tumour cell clones expressing high amounts of galectin-3 emerged with increasing levels of malignancy; and (iv) the level of accessible galectin-3-binding sites was apparently not heavily modified in the course of malignancy progression. In conclusion, the results obtained in the present study show that human astrocytic tumours are very heterogenous in their galectin-3 levels of expression. If high levels of galectin-3 determine the invasiveness potential of a tumour cell, then within a heterogenous tumour the presence of even a small, but actively proliferating number of tumour cell clones expressing high levels of galectin-3 can be expected to lead to tumour invasiveness.

The influence of L-triiodothyronine, L-thyroxine, estradiol-17beta, the luteinizing-hormone-releasing hormone, the epidermal growth factor and gastrin on cell proliferation in organ cultures of 35 benign and 13 malignant human thyroid tumors.

PURPOSE: To characterize the influence of six factors on human thyroid tissues at the cell-proliferation level. These six factors were the epidermal growth factor (EGF), the luteinizing-hormone-releasing hormone (LHRH), triiodothyronine, thyroxine, estradiol and gastrin. METHODS: Forty-eight human thyroid specimens were obtained from surgical resection and maintained alive for 48 h ex vivo (in vitro) under organotypic culture conditions. These specimens comprised 35 benign cases (17 multinodular goiters and 18 adenomas) and 13 cancers. Cell proliferation in the control and treated conditions (at a 5 nM dose) was assessed by means of the thymidine labeling index, which enables the percentage of cells in the S phase of the cell cycle to be determined in accordance with autoradiographic procedures. RESULTS: The results show that, of the six factors tested here, EGF significantly (P < 0.05 to P < 0.001) increased cell proliferation in the greatest number of cancers as compared to what happened with the remaining five. Each of these six factors significantly increased or decreased proliferative cell activity in some 10%-30% of the cases under study. CONCLUSIONS: Triiodothyronine, thyroxine, LHRH and gastrin may increase or decrease cell proliferation in human thyroid tissues, whether benign or malignant, to the same extent as other hormones and/or growth factors such as thyrotropin, EGF, insulin-like growth factor 1, transforming growth factor beta1 and estradiol the effects of which on thyroid cell proliferation are already well documented in the literature.

In vitro motility evaluation of aggregated cancer cells by means of automatic image processing.

BACKGROUND: Set up of an automatic image processing based method that enables the motility of in vitro aggregated cells to be evaluated for a number of hours. METHODS: Our biological model included the PC-3 human prostate cancer cell line growing as a monolayer on the bottom of Falcon plastic dishes containing conventional culture media. Our equipment consisted of an incubator, an inverted phase contrast microscope, a Charge Coupled Device (CCD) video camera, and a computer equipped with an image processing software developed in our laboratory. This computer-assisted microscope analysis of aggregated cells enables global cluster motility to be evaluated. This analysis also enables the trajectory of each cell to be isolated and parametrized within a given cluster or, indeed, the trajectories of individual cells outside a cluster. RESULTS: The results show that motility inside a PC-3 cluster is not restricted to slight motion due to cluster expansion, but rather consists of a marked cell movement within the cluster. CONCLUSIONS: The proposed equipment enables in vitro aggregated cell motility to be studied. This method can, therefore, be used in pharmacological studies in order to select anti-motility related compounds. The compounds selected by the equipment described could then be tested in vivo as potential anti-metastatic.

Supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas are characterized by a differential expression of S100 proteins.

The levels of expression of the S100A1, S100A2, S100A3, S100A4, S100A5, S100A6 and S100B proteins were immunohistochemically assayed and quantitatively determined in a series of 95 astrocytic tumors including 26 World Health Organization (WHO) grade I (pilocytic astrocytomas), 23 WHO grade II (astrocytomas), 25 WHO grade III (anaplastic astrocytomas) and 21 WHO grade IV (glioblastomas) cases. The level of the immunohistochemical expression of the S100 proteins was quantitatively determined in the solid tumor tissue (tumor mass). In addition twenty blood vessel walls and their corresponding perivascular tumor astrocytes were also immunohistochemically assayed for 10 cases chosen at random from each of the four histopathological groups. The data showed modifications in the level of S100A3 protein expression; these modifications clearly identified the pilocytic astrocytomas from WHO grade II-IV astrocytic tumors as a distinct biological group. Modifications in the level of S100A6 protein expression enabled a clear distinction to be made between low (WHO grade I and II) and high (WHO grade III and IV) grade astrocytic tumors. Very significant modifications occurred in the level of S100A1 protein expression (and, to a lesser extent, in their of the S100A4 and S100B proteins) in relation to the increasing levels of malignancy. While the S100A5 protein was significantly expressed in all the astrocytic tumors (but without any significant modifications in the levels of malignancy), the S100A2 protein was never expressed in these tumors. These data thus indicate that several S100 proteins play major biological roles in human astrocytic tumors.

Gastrin inhibits motility, decreases cell death levels and increases proliferation in human glioblastoma cell lines.

Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma. This invasion relates mainly to cell motility, which enables individual cell migration to take place. The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs. cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines. A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin. While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines. In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models. All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics.

Determination of growth fraction and cell density to evaluate the potential growth of human oligodendroglial and astrocytic tumours.

UNLABELLED: The object of this work was PURPOSE: to develop a methodology that enables net tumour growth, a balance between actual tumour growth and tumour cell loss, to be approximately evaluated. METHODS: The methodology proposed relies on detecting the growth fraction immunohistochemically by means of MIB-1 antibody labelling combined with cell density determination, carried out on 5-microm-thick Feulgen-stained histological sections with computer-assisted microscopy. The series investigated included 25 oligodendrogliomas (OLG-II), 9 anaplastic oligodendrogliomas (OLG-III). 13 astrocytomas (AST), 14 anaplastic astrocytomas (ANA) and 8 mixed oligoastrocytomas (OLG-AST). RESULTS: The results show that the biological characteristics of some cases were in total accordance with their histopathological diagnoses. This was the case for the "weakly proliferating weakly dense" OLG-II and AST-II tumours, and for the "highly proliferating highly dense" OLG-III and AST-III ones. In contrast, the biological characteristics of some cases seemed to contradict the histopathological case labels. This was the case for the "highly proliferating highly dense" OLG-II and AST-II tumours, the biological aggressiveness of which would be undervalued on the basis of the morphology-based grading system alone, and also for the "weakly proliferating weakly dense" OLG-III and AST-III tumours, the aggressiveness of which would be overvalued. CONCLUSIONS: Combining the determinations of the MIB-1 and the cell density variables appears to be satisfactory in terms of the cell kinetic characterization of glial tumours as a complement to the prognostic information given by a morphology-based grading system alone.

Characterization of astroglial versus oligodendroglial phenotypes in glioblastomas by means of quantitative morphonuclear variables generated by computer-assisted microscopy.

The current WHO classification places glioblastomas in the astrocytoma category. However, whether or not glioblastomas also show oligodendroglial differentiation remains a matter of controversy. This study investigates, at the morphonuclear level, the hypothesis that some glioblastomas (GBMs) may also represent the ultimate level of malignancy in the oligodendroglial lineage. Using a series of 164 GBMs, we sought to ascertain whether any of these GBMs exhibited phenotypical characteristics that were more closely related to oligodendroglial lineages than astrocytic lineages. Phenotypical features were quantitatively determined by means of the computer-assisted microscope analysis of Feulgen-stained nuclei, a process that made it possible to quantitatively describe the patterns of the cell nuclei (and, more specifically, of their chromatin) through 16 variables, and the distribution of the nuclear DNA content (DNA ploidy) through 8 variables. The phenotypical characteristics typical of astrocytic and oligodendroglial tumors were analyzed by means of Discriminant Analysis, a statistical multivariate analysis, performed on a series of 65 astrocytic and oligodendroglial tumors. This series consisted of 14 WHO grade II and 19 grade III astrocytomas and 24 WHO grade II and 8 grade III oligodendrogliomas. This multivariate analysis enabled an accurate model to be produced that distinguished between astrocytomas and oligodendrogliomas on the basis of 5 cytometry-generated variables. This model was used to characterize the phenotype of each of the 164 glioblastomas. The results show that of these 164 glioblastomas, 6 (about 3.5%) displayed phenotypes that were very similar to oligodendrogliomas, and 141 displayed phenotypes that were very similar to astrocytomas. The phenotypes of the 17 remaining GBMs were too ambiguous to be categorized as having a pure astrocytic or oligodendroglial lineage.

Direct relationship between hormone sensitivity level and growth pattern. Evidence in 18 gastrointestinal neoplastic cell lines.

OBJECTIVE: We investigated whether a relationship exists in terms of growth pattern and hormone sensitivity in 18 gastrointestinal neoplastic cell lines. Hormones studied included gastrin, epidermal growth factor, estradiol and luteinizing hormone-releasing hormone. STUDY DESIGN: The growth patterns were assessed by means of computer-assisted microscope analysis of Feulgen-stained nuclei combined with the mathematical Delaunay triangulation and Voronoi paving techniques. This methodology enabled four variables characterizing the cell colony patterns to be computed. The information contributed by these variables was analyzed by means of discriminant analysis and the decision tree technique. RESULTS: Each phenotype (sensitivity level) exhibited distinct growth pattern (or cell colony) characteristics in the case of each hormone and/or growth factor under study. Furthermore, the sensitivity of the gastrointestinal cell lines to a given hormone (or growth factor) appeared to be peculiar to the hormone (or growth factor). CONCLUSION: A direct relationship seems to exist between growth pattern and hormone sensitivity levels in gastrointestinal cancers, particularly colorectal.

Improving morphology-based malignancy grading schemes in astrocytic tumors by means of computer-assisted techniques.

We propose an original methodology which improves the accuracy of the prognostic values associated with conventional morphologically-based classifications in supratentorial astrocytic tumors in the adult. This methodology may well help neuropathologists, who must determine the aggressiveness of astrocytic tumors on the basis of morphological criteria. The proposed methodology comprises two distinct steps, i.e. i) the production of descriptive quantitative variables (related to DNA ploidy level and morphonuclear aspects) by means of computer-assisted microscopy and ii) data analysis based on an artificial intelligence-related method, i.e. the decision tree approach. Three prognostic problems were considered on a series of 250 astrocytic tumors including 39 astrocytomas (AST), 47 anaplastic astrocytomas (ANA) and 164 glioblastomas (GBM) identified in accordance with the WHO classification. These three problems concern i) variations in the aggressiveness level of the high-grade tumors (ANA and GBM), ii) the detection of the aggressive as opposed to the less aggressive low-grade astrocytomas (AST), and iii) the detection of the aggressive as opposed to the less aggressive anaplastic astrocytomas (ANA). Our results show that the proposed computer-aided methodology improves conventional prognosis based on conventional morphologically-based classifications. In particular, this methodology enables some reference points to be established on the biological continuum according to the sequence AST-->ANA-->GBM.