Dolores V. Dougherty

An ultrastructural and immunocytochemical analysis of leptomeningeal and meningioma cultures.

Using immunocytochemical methods, we localized several glycoproteins of the extracellular matrix to leptomeningeal cells and meningiomas in vitro. Three cell lines derived from normal human leptomeninges and seven from meningiomas were studied by indirect immunofluorescence to evaluate the cellular production of fibronectin, laminin, collagen type IV, and procollagen type III. All leptomeningeal cell lines stained intensely and uniformly for all matrix proteins; all meningioma cell cultures stained uniformly, but the intensity of staining varied considerably. After removal of the cells in culture adherent to glass with 25-mM ammonium hydroxide, indirect immunofluorescence demonstrated an exuberant residual extracellular residue enriched with fibronectin, laminin, collagen type IV, and procollagen type III. Electron microscopic examination of all leptomeningeal and meningioma cultures revealed desmosomes and dense tonofilament formation; in addition, granular, filamentous basement membrane-like material was abundant in the extracellular spaces of all cultures. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the cell layer of two leptomeningeal and four meningioma cultures showed production of interstitial collagen types I and III; diethylaminoethyl (DEAE)-cellulose chromatography of the medium demonstrated preferential production of procollagen type I. Our findings show conclusively that normal arachnoid cells in vitro synthesize several of the collagen subtypes and may be responsible for the “fibrous response” of the leptomeninges to trauma, infection, or infiltration by tumor. The similarities between leptomeningeal cells and meningiomas demonstrated by electron microscopy and by indirect immunofluorescence support the notion that meningiomas are derived from arachnoid cells. The localization of various mesenchymal glycoproteins within the intra- and extracellular spaces and the ubiquity of specialized intercellular junctions suggest that leptomeningeal cells in culture have the potential to behave like both stromal and epithelial cells.

Comparison of the cytotoxic activities of cisplatin and carboplatin against glioma cell lines at pharmacologically relevant drug exposures

Carboplatin has lower nephro- and neurotoxicities and better penetration into brain tissue than cisplatin. If carboplatin has comparable cytotoxicity against glioma cells, it might have a therapeutic advantage in the treatment of malignant gliomas. Using an assay of colony-forming efficiency, we compared the cytotoxicity of these two drugs in human glioma cell lines SF-126, SF-188, U87-MG, and U251-MG. The experiments were designed so that the product of in vitro drug concentration (C) and time (T) would encompass the same range of values as the C × T of the ultrafilterable platinum plasma fraction as determined by pharmacokinetic studies in man. The in vitro stability of the drugs was evaluated by measuring the cytotoxicity of aged drugs with a microculture tetrazolium assay. Cisplatin and carboplatin were both stable during the 2-h treatment. The cytotoxic activities of these drugs at clinically achievable levels of drug exposure were of the same order of magnitude. These results, in conjunction with the lower nephro- and neurotoxicities of carboplatin, the higher platinum levels in brain tissue after treatment with carboplatin, and the encouraging results of carboplatin in the clinical treatment of brain tumors that have been demonstrated in other studies, suggest that carboplatin might be preferable to cisplatin in the treatment of patients with malignant glioma.

Clonogenicity of multiple populations of human glioma cells in vitro sorted by DNA content

Malignant human gliomas have a highly variable distribution of cell nuclei, consisting of diploid and/or other populations in terms of nuclear DNA content. In order to study in vitro clonogenicity of each population, dissociated or cultured human glioma cells were stained with 20 μM/ml of Hoechst 33342 dye (which stains viable DNA with minimal cell kill), and were sorted sterile into separate populations, based on specific nuclear DNA content, for clonogenicity assay. The colony‐forming efficiency (CFE) of tumor cells plated immediately after disaggregation of the biopsy specimens ranged from 0.0044 to 0.149%, and the CFEs increased dramatically with successive passages (to 5 to 40%). The CFEs of the individual populations sorted according to DNA content were similar within individual tumors. These results suggest not only that malignant gliomas are composed of multiple populations in terms of DNA content, but also that each of these populations contain clonogenic cells. The morphologic structure of cells within and among colonies did not appear to relate to DNA content.

Potentials and possible pitfalls of human stem cell analysis

SummaryA clonogenic cell assay for malignant brain tumors that permits the evaluation of tumor cell sensitivity to BCNU and that correlates with patient response to BCNU has been developed. The potential for a stem cell analysis of human tumors has been demonstrated by studies of the reasons for clinical drug failure, tumor heterogeneity, and age-response relationships.The basic requirements of a stem cell assay include the ability to dissociate representative single cells from solid tumors, to optimize culture conditions, and to characterize the growth of colonies. Exposure of cells to a drug in vitro must be comparable to the in situ situation; possible significant differences between short-term and ‘continuous’ treatment methods are emphasized. Also discussed are criteria for in vitro sensitivity of cells, problems inherent in the ‘early’ evaluation of cultures (at the cell ‘cluster’ stage), and the effects of system errors, which if overcome should lead to the development of analytic methods with a maximum sensitivity and predictive value.

Development of a non-selecting, non-perturbing method to study human brain tumor cell invasion in murine brain

SummaryThe infiltrative nature of glial and some meningeal neoplasms is responsible for the failure of surgical removal and high recurrence rate of these tumors. Modeling of this processin vitro andin vivo will lead to a better understanding of the pathophysiology of this process and identify targets for novel therapy directed towards this phenotype. We present the results of the development and refinement of two model systems of tumor invasion: onein vitro barrier assay using the basement membrane extract Matrigel, and onein vivo where molecular detection of tumor cells allows single cell discrimination byin situ hybridization histochemistry. These techniques have strong correlations which validate their utility as measures of nervous system tumor invasion.

Comparison of in vitro Cloning Assays for Drug Sensitivity Testing of Human Brain Tumours

Three in vitro clonogenic assays were used to determine the sensitivity of an established human glioblastoma cell line (U251-MG) to five chemotherapeutic agents. The colony-forming efficiency of untreated culture was 0.695 +/- 0.170 in a monolayer assay with irradiated feeder cells, 0.018 +/- 0.006 in a low-O2 agar assay, and 0.049 +/- 0.021 in a two-layer agar system with nutrient-enriched medium (p less than 0.001). Comparison of the slope of the regression line for the dose-response curve and the interpolated ID90 for each drug showed that U251-MG was equally sensitive to aziridinylbenzoquinone and dianhydrogalactitol in all three assays. The sensitivity of this cell line to 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), cis-dichlorodiammineplatinum (II) (CDDP) and 9-hydroxy-2-N-methylellipticine (HME), however, varied depending on the assay used. In no instance did U251-MG show greater sensitivity (lower ID90 or steeper slope) in the low-O2 agar assay than in the other assays. BCNU and CDDP were least active in the monolayer assay, whereas HME showed both the lowest ID90 and steepest slope using this technique. We conclude that different in vitro tumour clonogenic assays show different colony-forming efficiencies for the same cell line and may show different responses to certain drugs. Identification of accurate predictive models of drug sensitivity will require correlative in vivo and in vitro studies.

Experimental basis for increasing the therapeutic index of carboplatin in brain tumor therapy by pretreatment with WR compounds

SummaryWe tested an experimental strategy to decrease the dose-limiting hematotoxicity of carboplatin without compromising its activity against brain tumors. The effect of pretreatment with WR-1065, a chemomodifier that penetrates brain poorly, on carboplatins cytotoxicity was evaluated in human hematopoietic granulocyte-monocyte progenitor cells and in three human glioblastoma cell lines. WR-1065 reduced bone marrow toxicity without decreasing carboplatins activity against glioblastoma cells. These results suggest that the therapeutic index of carboplatin might be increased in the treatment of malignant brain tumors.

Effect of pharmacologic doses of zinc on the therapeutic index of brain tumor chemotherapy with carmustine.

To evaluate the potential differential effect of pretreatment with pharmacologic doses of the trace element zinc on the chemosensitivity of glioma cells and bone marrow cells for carmustine (BCNU), we performed in vitro and in vivo studies of zinc toxicity as well as of the combined treatment with zinc and the anticancer drug. We studied the in vitro effects on established human and rat glioma cell lines using a microcolorimetric growth assay and on murine bone marrow using a clonogenic assay for committed progenitor cells of the granulocyte-monocyte lineage. Zinc exposures of up to 100 μM for 120 h did not influence the growth of six of seven human glioma cell lines. Only U87MG demonstrated statistically significant toxicity during high zinc exposure (100 μM over 120 h). Dose-response growth curves generated for BCNU did not show protection against the anticancer agents by a 48-h pretreatment with different zinc concentrations. The clonogenic capacity of bone marrow cells was slightly reduced by in vitro culture for 24 and 48 h. Although this effect appeared to be more prominent in the presence of zinc supplementation, overall a statistically significant inhibition was seen only after exposure to a concentration of 100 μM zinc over 48 h. As compared with chemotherapy alone, in vitro pretreatment with 50 μM zinc over 48 h followed by chemotherapy resulted in an increased number of colony-forming unit-granulocyte monocyte (CFU-GM): CFU-GM increased by a factor of 2 for BCNU (60 μM×2 h). This statistically significant in vitro chemoprotection would translate into a dose-protection factor of 1.5, i.e., for the same level of myelosuppression, zinc pretreatment would allow administration of a 50% increased dose of BCNU. The in vivo studies were performed in an s.c. xenograft model of the human glioma cell line U87MG in athymic mice. The maximal tolerable pretreatment with zinc was determined to be a 10-day course of daily i.p. injections of 10 mg/kg ZnCl2. The subsequent i.p. administration of the dose lethal to 10% of the mice (LD10) and of a 1.5×LD10 dose of BCNU resulted in less bone marrow toxicity in pretreated animals than in non-zinc-pretreated mice as determined in a CFU-GM assay. Glioma colony-forming efficiency (CFE) assays, on the other hand, did not show any zinc-related difference in the BCNU sensitivity of U87MG. Our in vitro and in vivo results suggest the potential usefulness of high-zinc pretreatment for improving the therapeutic index of BCNU chemotherapy for gliomas.

Resistance of Brain Tumours to Chemotherapy: Preliminary Studies of Rat and Human Tumours *

There are several possible reasons why malignant brain tumours do not respond to chemotherapy. First, because of impaired drug delivery, chemotherapeutic agents might not reach all tumour cells in cytoxic concentrations. Second, tumour growth kinetics might affect the response to drugs because tumour cells are in an insensitive phase of the cell cycle when the drug is administered (10). Finally, tumour cells may survive therapy because of “inherent” cellular resistance.

Variable response to 1,3-bis(2-chloroethyl)-1-nitrosourea of human glioma cells sorted according to DNA content.

SummaryMalignant gliomas, especially glioblastoma multiforme, are composed of a considerable number of cells undergoing DNA synthesis and cells that contain various amounts of DNA. We have characterized cells dissociated from biopsy specimens of such tumors by sorting vital cell populations on the basis of DNA content and have determined the sensitivity of sorted cells to BCNU. Cultured human glioma cells from seven malignant gliomas were stained with Hoechst 33342 dye; the colony-forming efficiency (CFE) of stained tumor cells ranged from 2–50%. Stained cells were sorted on a fluorescence-activated cell sorter (FAGS-III). The surviving fraction of each population was determined after exposure to graded doses (2.5–10 μg/ ml × 2 h) of BCNU. Sorted populations of cells from 5 tumors had dose-response curves that were similar, although differences in cell kill of up to a half-log were commonly found between cells from different DNA peaks treated with the same BCNU dose. For two tumors, cells from the first peak (smallest DNA content) had distinct BCNU sensitivity compared to cells from the second and third peaks (largest DNA content); compared to other tumors in the series, cell kill differences were significant and greater than 1 log in magnitude for one anaplastic astrocytoma. This lack of uniformity in the response of cells within a tumor demonstrates the problem imposed by heterogeneity with regard to the interpretation of chemosensitivity testing of all cells within a single tumor.