A technique to analyse the emerging zonality of heterogeneous solid tumours

A technique to analyse the time-dependent emergence of homogeneous regions composed of cells from a single clone within an artificial (clonally) heterogeneous tumour is described. Neoplasms were grown in vivo as xenografts made from varying proportions of the dichotomous subpopulations (Clones A and D). They were sampled frequently for volume and composition. A variable number of tumour cross-sections were taken as part of the sampling technique. The random subsamples obtained from each cross-section were enzymatically disaggregated into single cells. From the single cell disaggregates the composition of the tumours was estimated. An estimator for the global proportion of cells was then calculated from all the single cell disaggregates. Time-dependent changes in the overall composition of the tumour requires that a time-dependent estimate of the global proportions of the subpopulations be calculated from each sample. Analysis of the sample proportions results in a statistic which can be tested for goodness-of-fit against a standardized normal variate as a test of emerging zonality. Data from three artificial admixtures were examined. The results show that 'zonality', i.e. regions composed primarily of single subpopulations, emerges in all cases. However, the rate at which the zones emerge appears to depend on the 'compositional stability'. Robustness studies show that the technique is robust with respect to the global estimator of the proportion.     

A technique to analyse the emerging zonality of heterogeneous solid tumours

Abstract A technique to analyse the time-dependent emergence of homogeneous regions composed of cells from a single clone within an artificial (clonally) heterogeneous tumour is described. Neoplasms were grown in vivo as xenografts made from varying proportions of the dichotomous subpopulations (Clones A and D). They were sampled frequently for volume and composition. A variable number of tumour cross-sections were taken as part of the sampling technique. The random subsamples obtained from each cross-section were enzymatically disaggregated into single cells. From the single cell disaggregates the composition of the tumours was estimated. An estimator for the global proportion of cells was then calculated from all the single cell disaggregates. Time-dependent changes in the overall composition of the tumour requires that a time-dependent estimate of the global proportions of the subpopulations be calculated from each sample. Analysis of the sample proportions results in a statistic which can be tested for goodness-of-fit against a standardized normal variate as a test of emerging zonality.
Data from three artificial admixtures were examined. The results show that 'zonality', i.e. regions composed primarily of single subpopulations, emerges in all cases. However, the rate at which the zones emerge appears to depend on the 'compositional stability'.
Robustness studies show that the technique is robust with respect to the global estimator of the proportion.     

Matrices with compliance comparable to that of brain tissue select neuronal over glial growth in mixed cortical cultures

Cortical neurons and astrocytes respond strongly to changes in matrix rigidity when cultured on flexible substrates. In this study, existing polyacrylamide gel polymerization methods were modified into a novel method for making substrates capable of engaging specific cell-adhesion receptors. Embryonic cortical dissociations were cultured on polyacrylamide or fibrin gel scaffolds of varying compliance. On soft gels, astrocytes do not spread and have disorganized F-actin compared to the cytoskeletons of astrocytes on hard surfaces. Neurons, however, extend long neurites and polymerize actin filaments on both soft and hard gels. Compared to tissue culture plastic or stiff gel substrates coated with laminin, on which astrocytes overgrow neurons in mixed cultures, laminin-coated soft gels encourage attachment and growth of neurons while suppressing astrocyte growth. The number of astrocytes on soft gels is lower than on hard even in the absence of mitotic inhibitors normally used to temper the astrocyte population. Dissociated embryonic rat cortices grown on flexible fibrin gels, a biomaterial with potential use as an implant material, display a similar mechano-dependent difference in cell population. The stiffness of materials required for optimal neuronal growth, characterized by an elastic modulus of several hundred Pa, is in the range measured for intact rat brain. Together, these data emphasize the potential importance of material substrate stiffness as a design feature in the next generation of biomaterials intended to promote neuronal regeneration across a lesion in the central nervous system while simultaneously minimizing the ingrowth of astrocytes into the lesion area.     

Monoclonal antibodies to heterogeneous nuclear RNA-protein complexes. The core proteins comprise a conserved group of related polypeptides

Hybridomas secreting monoclonal antibodies that react with heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins have been isolated by immunizing BALB/c mice with RNP particles isolated from chicken and screening the fusion products with mouse RNP complexes. The antibodies show varying affinities for the hnRNP core proteins that have been blotted onto nitrocellulose. The majority of the immunoglobulins react with all the core group proteins although several recognize subsets of the hnRNP polypeptides. The clones are specific for different antigenic determinants as shown by their inability to compete with one another for binding sites. A mild proteolytic digestion of hnRNP proteins generates fragments that have uniformly lost 12 kDa and contain the antigenic determinants recognized by several of the monoclonal antibodies. Thus, it appears the core proteins comprise a family of related polypeptides possessing underlying structural similarities. Polypeptides similar in number and molecular weights that have antigenic determinants cross-reactive with those of mouse RNP have been found in a number of organisms, thereby emphasizing their possible common structure and function in higher eukaryotes. No difference in the distribution within the cell of individual or groups of core proteins has so far been detected by indirect immunofluorescence.     

Expression of neuronal and glial markers in so-called oligodendroglial tumors induced by transplacental administration of ethyl-nitrosourea in the rat.

A series of 18 tumors with histological features of oligodendrogliomas, induced in the rat by means of transplacental ethyl-nitrosourea administration were studied for immunohistochemical demonstration of neuronal (synaptophysin and neurofilament protein) and glial (gliofibrillar acidic protein and vimentin) markers. Most of the tumors showed cells with strong positivity to synaptophysin and to a lesser degree, to neurofilament protein, suggesting the neuronal character of these neoplasms. In 10 tumors, cells with strong positivity to vimentin were found, and in three cases, tumoral cells expressed gliofibrillar acidic protein. The observation that ENU-induced oligodendroglial tumors express neuronal and, to a minor degree, glial markers, suggests their interpretation as primitive neuroectodermal tumors with clear neuronal differentiation.     

Neuroimaging biomarkers for epilepsy: Advances and relevance to glial cells

Glial cells play an important role in normal brain function and emerging evidence would suggest that their dysfunction may be responsible for some epileptic disease states. Neuroimaging of glial cells is desirable, but there are no clear methods to assess neither their function nor localization. Magnetic resonance imaging (MRI) is now part of a standardized epilepsy imaging protocol to assess patients. Structural volumetric and T2-weighted imaging changes can assist in making a positive diagnosis in a majority of patients. The alterations reported in structural and T2 imaging is predominately thought to reflect early neuronal loss followed by glial hypertrophy. MR spectroscopy for myo-inositol is a being pursued to identify glial alterations along with neuronal markers. Diffusion weighted imaging (DWI) is ideal for acute epileptiform events, but is not sensitive to either glial cells or neuronal long-term changes found in epilepsy. However, DWI variants such as diffusion tensor imaging or q-space imaging may shed additional light on aberrant glial function in the future. The sensitivity and specificity of PET radioligands, including those targeting glial cells (translocator protein) hold promise in being able to image glial cells. As the role of glial function/dysfunction in epilepsy becomes more apparent neuroimaging methods will evolve to assist the clinician and researcher in visualizing their location and function.     

Brain postsynaptic densities: the relationship to glial and neuronal filaments

Preparations of isolated brain postsynaptic densities (PSDs) contain a characteristic set of proteins among which the most prominent has a molecular weight of approximately 50,000. Following the suggestion that this major PSD protein might be related to a similarly sized component of neurofilaments (F. Blomberg et al., 1977, J. Cell Biol., 74:214- 225), we searched for evidence of neurofilament proteins among the PSD polypeptides. This was done with a novel technique for detecting protein antigens in SDS-polyacrylamide gels (immunoblotting) and an antiserum that was selective for neurofilaments in immunohistochemical tests. As a control, an antiserum against glial filament protein (GFAP) was used because antisera against GFAP stain only glial cells in immunohistochemical tests. They would, therefore, not be expected to react with PSDs that occur only in neurons. The results of these experiments suggested that PSDs contain both neuronal and also glial filament proteins at higher concentrations than either synaptic plasma membranes, myelin, or myelinated axons. However, immunoperoxidase staining of histological sections with the same two antisera gave contradictory results, indicating that PSDs in intact brain tissue contain neither neuronal or glial filament proteins. This suggested that the intermediate filament proteins present in isolated PSD preparations were contaminants. To test this possibility, the proteins of isolated brain intermediate filaments were labeled with 125I and added to brain tissue at the start of a subcellular fractionation schedule. The results of this experiment confirmed that both neuronal and glial filament proteins stick selectively to PSDs during the isolation procedure. The stickiness of PSDs for brain cytoplasmic proteins indicates that biochemical analysis of subcellular fractions is insufficient to establish a given protein as a synaptic junctional component. An immunohistochemical localization of PSDs in intact tissue, which has now been achieved for tubulin, phosphoprotein I, and calmodulin, appears to be an essential accessory item of evidence. Our findings also corroborate recent evidence which suggests that isolated preparations of brain intermediate filaments contain both neuronal and glial filaments.     

Monoclonal antibodies against group A streptococcal polysaccharide reacting with basal-cell antigen of tumors histogenetically related to epidermal tissues

Immunofluorescence of tumour tissues histogenetically related to tissues containing cross-reacting squamous epithelial basal-cell antigen (BCAg) was performed using monoclonal antibodies (McAb) A6/I-D to squamous epithelial basal-cell antigen cross-reacting with group A staphylococcal polysaccharide. BCAg was found in tumour cells arising from surface tissues of the epidermal type (basal-cell cancer, squamous-cell cancer of the skin, esophagus, cervix uteri and other organs) and was not found in glandular tumours (adenocarcinoma of the stomach, intestine, mammary glands). The results obtained indicate that McAb A6/I-D against cross-reacting BCAg may be used while characterizing the majority of normal epidermal tissues and tumours arising from tissues containing cross-reacting BCAg.     

Degradation of the neuropeptide somatostatin by cultivated neuronal and glial cells.

The enzymatic degradation of the neuropeptide somatostatin was investigated in cultivated cells and subcellular fractions from rat brain. Dissociated neurones, astrocytes, and oligodendrocytes obtained from rat cerebral cortex were of more than 85-98% purity as evidenced by immunostaining with antisera to cell specific markers. All of these cell types were able to cleave radiolabeled somatostatin to smaller fragments, especially cultivated astrocytes with the highest specific activity. The neuroblastoma cell line N1E-115 did not measureably cleave somatostatin. The somatostatin-degrading proteases of the cultivated brain cells could be differentiated by their sensitivity to protease inhibitors and by the fragments produced: astrocytes contain a metallo-endoprotease sensitive to phenanthroline which cleaves somatostatin at the Phe6-Phe7 and Thr10-Phe11 bonds, whereas the endoprotease(s) of neurones and oligodendrocytes was insensitive to chelating agents but strongly inhibited by the antibiotic bacitracin. In accordance with this, the bacitracin-sensitive activity was mainly recovered in the synaptic plasma membrane and myelin subcellular fractions obtained by differential centrifugation of rat cerebral cortex homogenate. However, the highest total and specific somatostatin-degrading activity was detected in the cytosolic fraction.     

A cysteine proteinase secreted from human breast tumours is immunologically related to cathepsin B.

The stable cathepsin B-like cysteine (thiol) proteinase secreted from human breast tumours in culture was shown to be destabilized by mercurial compounds. After such treatment the enzyme cross-reacts in a radioimmunoassay with a monospecific antiserum to human liver cathepsin B. It is suggested that the secreted enzyme may be a precursor form of lysosomal cathepsin B.     

Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity

Huntington disease (HD) is characterized by the preferential loss of striatal medium-sized spiny neurons (MSNs) in the brain. Because MSNs receive abundant glutamatergic input, their vulnerability to excitotoxicity may be largely influenced by the capacity of glial cells to remove extracellular glutamate. However, little is known about the role of glia in HD neuropathology. Here, we report that mutant huntingtin accumulates in glial nuclei in HD brains and decreases the expression of glutamate transporters. As a result, mutant huntingtin (htt) reduces glutamate uptake in cultured astrocytes and HD mouse brains. In a neuron-glia coculture system, wild-type glial cells protected neurons against mutant htt-mediated neurotoxicity, whereas glial cells expressing mutant htt increased neuronal vulnerability. Mutant htt in cultured astrocytes decreased their protection of neurons against glutamate excitotoxicity. These findings suggest that decreased glutamate uptake caused by glial mutant htt may critically contribute to neuronal excitotoxicity in HD.     

Accumulation of sulfatide in neuronal and glial cells of arylsulfatase A deficient mice

Arylsulfatase A (ASA) degrades sulfatide, seminolipid and lactosylceramide sulfate, glycolipids recognized by the Sulph I antibody although sulfatide is considered the main antigen. Sulfatide is myelin associated but studies have shown a minor distribution also in non-myelin forming cells. The aim of this work was to further study sulfatide in neurons and astrocytes by immunohistochemistry, facilitated by investigation of tissue from adult ASA deficient (ASA ?/?) mice. Cells with a low presence of sulfatide might be detected due to lack of ASA activity and accumulation of Sulph I antigens. Sulfatide positive astrocytes and neurons were more numerous and intensely stained in ASA ?/? mice, demonstrating a sulfatide accumulation compared to controls. Sulph I staining was especially increased in the molecular layer of cerebellum, in which Purkinje cell dendrites displayed an altered morphology, and in layer IV–VI of cerebral cortex. In hippocampus, immunostaining was found in neuronal cytoplasm in ASA ?/? but in nuclear membranes of control mice. We observed a gray matter astrogliosis, which appeared to be associated to sulfatide accumulation. In addition, the developmental change (<20 months) of Sulph I antigens, galactosylceramide, phospholipids and cholesterol were followed by lipid analyses which verified sulfatide and seminolipid accumulation in adult ASA ?/? mice, although no lactosylceramide sulfate could be detected. In addition to demonstrating sulfatide in neurons and astrocytes, this study supports the value of ASA ?/? mice as a model for metachromatic leukodystrophy and suggests that accumulation of sulfatide beyond myelin might contribute to the pathology of this disease.     

Monoclonal antibody (M2) to glial and neuronal cell surfaces

A monoclonal antibody designated M2 arose from the fusion of mouse myeloma cells with splenocytes from a rat immunized with particulate fraction from early postnatal mouse cerebellum. Expression of M2 antigen was examined by indirect immunofluorescence on frozen sections of developing and adult mouse cerebellum and on monolayer cultures of early postnatal mouse cerebellar cells. In adult cerebellum, M2 staining outlines the cell bodies of granule and Purkinje cells. A weaker, more diffuse staining is seen in the molecular layer and white matter. In sections of newborn cerebellum, M2 antigen is weakly detectable surrounding cells of the external granular layer and Purkinje cells. The expression of M2 antigen increases during development in both cell types, reaching adult levels by postnatal day 14. At all stages of postnatal cerebellar development, granule cells that have completed migration to the internal granule layer are more heavily stained by M2 antibodies than are those before and in process of migration. In monolayer cultures, M2 antigen is detected on the cell surface Of all GFA protein-positive astrocytes and on more immature oligodendrocytes, that express 04 antigen but not 01 antigen. After 3 days in culture, tetanus toxinpositive neurons begin to express M2 antigen. The same delayed expression of M2 antigen on neurons is observed in cultures derived from mice ranging in age from postnatal day 0 to 10.     

S100 beta stimulates calcium fluxes in glial and neuronal cells.

The glial-derived protein S100 beta can act as a mitogen or a neurotrophic factor, stimulating proliferation of glial cells or differentiation of immature neurons. We report here that dimeric S100 beta evokes increases in intracellular free calcium concentrations ([Ca2+]i) in both glial cells and neuronal cells. The [Ca2+]i increase exhibited a rapid transient component which was not affected by removal of extracellular calcium and a sustained component which appeared to require influx of extracellular calcium through Ni(2+)-sensitive channels. S100 beta also stimulated hydrolysis of phosphoinositides, suggesting a mobilization of calcium from intracellular stores. These data suggest that although the final biological responses of neuronal and glial cells to S100 beta are different, transduction of the S100 beta signal in both cell types involves changes in [Ca2+]i.