Cell-lines derived from human gliomas activate multiple autocrine pathways.

Fifteen permanent cell lines derived from maligant human gliomas and 5 sublines derived thereof were analyzed for the secretion of mitogenic activities. Conditioned media (c-med) from all cells were obtained from four to six day long periods of serum-free culture. The optimal conditioning time period resulting in maximal activity of the media varied between the cell lines. These conditioned media were added in final concentrations ranging from 5% to 60% to autologous and heterologous glioma cell cultures in serum-free conditions. These cultures were counted after 2, 4 and 6 days. It was observed, that there were two distinct groups of cells in respect to the response to the autologous media. 11 cell lines responded to their own c-med with dose dependent, enhanced rate of proliferation. The other cell lines proliferated in serum-free medium without response to the addition of c-med. Analyzing the responses to heterologous assays it was found, that media which did not show effect in the homologous system, were mitogenic for other cell lines. Also, cells which did not respond to their own media, responded to heterologous c-med. This study indicates, that in vitro two groups of glioma cell-lines can be distinguished in respect to the response pattern to autologous growth factors secretion. It was also seen, that the activities supporting proliferation did not act on five randomly chosen meningiomas in early culture, indicating some degree of lineage specificity. From this study it may be concluded that active growth factor secretion by tumor cells does not necessarily imply that the cells depend on such mechanism for maintenance of cell division, Furthermore, it needs to be acknowledged, that there may be a multiplicity of autocrine/paracrine growth factors secreted by individual cell lines and possibly tumors.

Cyst fluids of malignant human brain tumors contain substances that stimulate the growth of cultured human gliomas of various histological type.

The contents of 14 cysts that were located within human intracranial tumors were obtained at surgery by needle aspiration. These tumor cyst fluids (TCFs) were mostly derived from glial tumors (10 cases). TCFs from one metastasis from a mammary carcinoma, one cystic meningioma, one hemangioblastoma, and a cystic acoustic neurinoma were also included. These TCFs were added to primary cultures of human gliomas, established human glioma cell lines, and normal human arachnoid cells in culture. The presence of proliferation-promoting factors in all cyst fluids could be demonstrated. On the basis of the response patterns of the cultures, it was possible to distinguish different levels of growth autonomy and growth factor sensitivity among these cultures and to speculate about varying degrees of cellular autocrine activation. The TCFs appear to contain factors that are not normally present in fetal calf serum, which is a regular constituent of most cell culture media. Some primary cultured cells as well as cell lines react in an oversensitive manner to the addition of TCFs.

Culture of human brain tumors on an extracellular matrix derived from bovine corneal endothelial cells and cultured human glioma cells.

Cell culture has become an integral part of the daily routine of most oncology laboratories. It has enabled researchers to investigate a wide range of cellular parameters in a defined system in which the experimental conditions can be controlled and repeated. Although the manufacturers of tissue culture materials are continually improving their products, cell attachment and initial survival of primary cultures from tumor cells are still problems in many laboratories. Many different approaches have been taken to circumvent this problem, and coating of tissue culture dishes with attachment enhancers, such as polyamino acids (1), fibronectin (2), laminin (3), and collagen (4), has been found helpful. For a long time, it was known that endothelial cells produce a basement membrane, and this led to the use of bovine corneal basement membrane by Gospodarowicz et al. (5,6), in their research into the phenomenon of regeneration and nonregeneration of corneal endothelium in different species. The application of this bovine corneal extracellular matrix (bECM) has since been greatly expanded (5,6). bECM has found broad approval, and has been used for mammary carcinoma (7), urological tumors (8), and different kinds of pituitary adenomas (9,10) as well as CNS tumors (11).

Cell culture of human brain tumors on extracellular matrices : methodology and biological applications.

Cell culture is one of the major tools of cell biologists. It has also become an integral part of the daily routine of most oncology laboratories for the purpose of karyotyping, chemoresistance testing, or basic research. It provides investigators with an opportunity to investigate many cellular parameters and interactions in an in vitro system in which the experimental conditions can be controlled and repeated With many tissues, either human or animal, the problems of cell culture are cell attachment and initial survival. Particularly the primary cultures derived from tumor specimens are a problem in many laboratories. Apart from modifications in the composition of tissue-culture plastic materials, other approaches have been used to get around this problem, such as coating of tissue-culture dishes with attachment enhancers, such as polyamino acids (1), fibronectin (2), laminin (3), and collagen (4) Since it was known that endothelial cells are capable of producing a basement membrane even in vitro, bovine cornea1 endothelial basement membrane was explored by Gospodarowicz et al. for its role in regeneration and nonregeneration of cornea1 endothelium in different species This bovine cornea1 extracellular matrix (bECM) was found useful in the cell culture of a wide range of different cells (5, 6), and bECM as well as other ECMs were employed in the cell biology of tumor cells derived from mammary carcinomas (7), urological tumors (8), and different kinds of pituitary adenomas (9, 10), as well as CNS tumors (11), which is the topic of this chapter.

Antigenic staining patterns of human glioma cultures: primary cultures, long-term cultures and cell lines.

The immunocytochemical staining patterns of cultured glioma cells were investigated. Fifty nine individual cases were stained at different in vitro ages for glial fibrillary acidic protein, fibronectin, galactocerebroside, HNK-1/Leu 7, A2B5, vimentin, factor VIII and A4. Histologically, the cases were composed of eight low-grade astrocytomas, 11 high-grade astrocytomas, four low-grade oligodendrogliomas, seven high-grade oligodendrogliomas and 29 glioblastomas. The 45 cases were analysed within the first 3 weeks of culture, many of them as primary cultures. In 11 cases stainings were performed repeatedly at intervals of up to 6 months. Glial fibrillary acidic protein staining was positive in most of the early cultures of astrocytomas (low and high grade) and glioblastomas; expression in more than 50% of the cells was found in 1 of 5 low-grade astrocytomas, 5 of 11 high-grade astrocytomas and 14 of 29 glioblastomas. Two of the high-grade astrocytomas were stained once more after 6 weeks in culture and were found to be only 1% positive for glial fibrillary acidic protein but strongly positive for fibronectin. The same was true for five of the glioblastoma cases. Two of these cases remained glial fibrillary acid protein positive and developed into stable permanent cell lines. Only one case started with 1% of glial fibrillary acidic protein positive cells and later developed into a 99% glial fibrillary acidic protein positive cell line. Neither HNK-1/Leu 7 expression nor A2B5 staining appeared to have a relationship to the glial fibrillary acidic protein staining. It was observed that glial fibrillary acidic protein and HNK-1/Leu 7 were both 100% in some cases but that later one of the two antigens disappeared but not the other. The amount of glial fibrillary acidic protein staining does not allow the prediction of A2B5 staining. The study shows that initiation of primary cultures on an extracellular matrix yields more glial fibrillary acidic protein positive cells in primary cultures than have been found in other studies. It is concluded that only a rigid standardization of culture conditions will ensure the validity of comparisons of in vitro data obtained in primary cultures.

Human neurocytoma cells in culture show characteristics of astroglial differentiation.

Cultured human neurocytoma cells from two neurosurgical patients were analysed for their immunocytochemical staining patterns and growth characteristics. In both cases, the cells stained positive for glial acidic fibrillary protein (GFAP) within one day of tissue culture in medium, with and without fetal calf serum, whereas the histological tumor specimens were negative. Both cases contained cells concomitantly expressing GFAP and synaptophysin (SNP) in the primary cultures. Epidermal growth factor (EGF) was mitogenic for the cultured cells but not platelet derived growth factor alpha (PDGF AA) or nerve growth factor (NGF). It is concluded that the human neurocytomas may represent neoplasms of a pluripotent neuroglial cell which can provide an interesting model to study the determinants for human glial/neuronal differentiation in vitro.

Glioma biology in vitro: goals and concepts.

Gliomas are heterogeneous in their cellular composition, affecting therapeutic efforts such as surgical removal, radiotherapy, chemotherapy and immunotherapy. 106 gliomas were taken into culture in our laboratory and 12 cell lines could be established there from. Experiments were carried out in as many early cultures as possible and with the constant experimental system of the cell lines. To subdivide and possibly classify the heterogeneous group of gliomas the following approaches emerged: Immunostaining of cells for glial markers such as GFAP, A4, A2B5, Leu 7 as well as fibronectin will allow one to distinguish different groups of glial cultures. Performance of growth factor sensitivity tests allows the assessment of major aspects of growth control in cultured gliomas. Cytogenetic evaluation in early cultures and correlation with the expression of oncogenes may yield useful information on mechanisms of escape from normal growth control. One of our cell lines (NCE-G28) in which cells switch from GFAP to fibronectin expression and transiently express the x-hapten may serve as a model to study crucial aspects of cellular differentiation. Using different extracellular matrices for the initiation of cultures even from very benign lesions with low proliferative potential it is possible to include these into comparative studies with glioblastomas.