Evolution of the brain tumour spheroid model: transcending current model limitations.

Tumour recurrence and the high mortality and morbidity associated with malignant brain tumours may be attributed to the failure of current therapeutic modalities (surgery, radiation and chemotherapy) to control the invasion of malignant brain tumour cells into healthy brain tissue. Several in vitro and in vivo models have been developed and used to study brain tumour invasion and cell motility. Here, we review some of the traditional in vitro models of brain tumour invasion and the latest adaptations to the widely used spheroid model. Several research groups studying the mechanisms mediating brain tumour invasion have made important contributions to the field by improving in vitro models of tumour migration and invasion. Sharing these advances will hopefully accelerate experimental discovery and the development of novel anti-invasion brain tumour therapies.

Adaptive response in patients treated with 131I.

UNLABELLED: The aim of this study was to investigate whether an adaptive response (defined as the induction of radiation tolerance after a small dose of radiation) could be observed in peripheral blood lymphocytes of patients treated with 1311 for thyroid disease. METHODS: For each patient, blood samples were taken immediately before and 1 wk after 131I administration. Each blood sample was divided into 3 fractions and the fractions were subsequently irradiated in vitro with 0, 0.5, and 1.0 Gy 60Co gamma-rays. After blood culture for 70 h, cells were harvested and stained with Romanowsky-Giemsa and micronuclei were counted in 1000 binucleated cells. The increase in micronuclei by the in vitro irradiation of the blood samples taken before and after therapy was compared. In this setup, an adaptive response is represented by a significant decrease of the in vitro induced micronucleus yield after therapy compared with that before therapy. The iodine therapy can be considered as an in vivo adaptation dose, after which the subsequent in vitro irradiation acts as a challenge dose. To investigate the reproducibility of the method, 2 subsequent blood samples of healthy volunteers were taken 7 d apart. Irradiation and cell culture were performed as described. RESULTS: In 8 of 20 patients, a significant (P = 0.0002) decrease was found in the in vitro induced micronucleus yield in the blood sample taken 1 wk after 1311 administration compared with that of the blood sample taken before therapy. No significant (P > 0.1) differences were observed between these 8 patients and the other patients when the number of micronuclei induced in vivo by the iodine treatment and the resulting equivalent total body dose were compared. None of the control subjects showed a significant change in micronucleus yield after in vitro irradiation between both blood samples taken 1 wk apart. CONCLUSION: The iodine treatment can act as an in vivo adaptation dose and can induce an adaptive response that is observed by a decrease of the cytogenetic damage in peripheral blood lymphocytes after in vitro irradiation as a challenge dose. A large interindividual difference was observed.

Estimation of risk based on biological dosimetry for patients treated with radioiodine.

A multicentre study was undertaken to assess the cytogenetic damage to peripheral blood lymphocytes in 31 patients treated with 131I for thyrotoxicosis using the cytokinesis-blocked micronucleus assay. The results were compared to those for eight thyroid carcinoma patients using the same method. For each patient, blood samples were taken immediately before and 1 week after iodine administration. The first blood sample was divided into three fractions and each fraction was subsequently irradiated in vitro with 0, 0.5 and 1 Gy 60Co gamma rays, respectively. After blood culture for 70 h, cells were harvested, stained with Romanowsky-Giemsa and the micronuclei scored in 1000 binucleated cells. For both patient groups, a linear-quadratic dose-response curve was fitted through the data set of the first blood sample by a least squares analysis. The mean increase in micronuclei after 131I therapy (second blood sample) was fitted to this curve and the mean equivalent total body dose (ETBD) calculated. Surprisingly, in view of the large difference in administered activity between thyroid carcinoma patients and thyrotoxicosis patients, the increase in micronuclei after therapy (mean +/- S.D.: 32 +/- 30 and 32 +/- 23, respectively) and the equivalent total body dose (0.34 and 0.32 Gy, respectively) were not significantly different (P > 0.1). The small number of micronuclei induced by 131I therapy (32 +/- 29), compared with external beam radiotherapy for Hodgkin's disease (640 +/- 381) or cervix carcinoma (298 +/- 76) [1], gave a cancer mortality estimate of less than 1%. This also explains why late detrimental effects in patients after 131I treatment have not been reported in the literature.

Failure of nimodipine to prevent brain damage in a global brain ischemia model in the rat.

In view of our negative results with the calcium antagonist nimodipine as a cerebroprotective agent in a cardiopulmonary resuscitation model in the rat, we examined the protective effects of nimodipine in the four-vessel (carotid and vertebral) occlusion model, a model of global brain ischemia without important cardiovascular depression. Survival and neurological status were monitored and after 72 h the hippocampus was resected and examined for histological evaluation. The animals were treated blindly and randomly with either nimodipine, its solvent or saline given subcutaneously. In two separate studies, high doses (total dose: 5 mg/kg) or low doses of nimodipine (total dose: 1.6 mg/kg) were administered. In the high dose series, the survival rates at 72 h in the nimodipine group, the saline group and the solvent group were 4% (2/44), 19% (7/37) and 20% (8/41), respectively; in the low dose series, the figures were 26% (13/50), 34% (15/44) and 39% (18/46), respectively. The differences between nimodipine, solvent and saline were not statistically significant. Likewise, no differences in neurological status or histological brain damage were found. These data suggest that nimodipine offers no cerebral protection in global brain ischemia and may even be toxic, especially when given in high doses.

Lymphocyte labeling with technetium-99m-HMPAO: a radiotoxicity study using the micronucleus assay.

The cytogenetic radiation damage to lymphocytes after in-vitro labeling of mixed leukocytes and isolated lymphocytes with 99mTc-hexamethylpropyleneamine oxime (HMPAO) was evaluated using the cytokinesis-blocked micronucleus assay. A direct assessment of the radiation damage to the lymphocytes after a labeling procedure of leukocytes separated from 46 ml blood with 740 MBq of 99mTc-HMPAO was not possible due to an almost complete impairment of the proliferative capacity. By starting with isolated lymphocytes, the number of micronuclei was studied versus the intracellular activity concentration in the range 0-3 MBq/10(7) lymphocytes for three donors. A comparison of these results with the dose response of the micronucleus incidence in lymphocytes after in-vitro irradiation with x-rays allowed an individual assessment of the x-ray dose, inducing the equivalent amount of clastogenic damage as the intracellular activity after 99mTc-HMPAO labeling. Based on an extrapolation of these data, the radiation damage of the lymphocytes due to self-irradiation in a labeling procedure of leukocytes with 740 MBq of 99mTc-HMPAO was estimated to be equivalent to 26 Gy of x-rays. Due to the observed almost complete inhibition of the proliferative capacity at this high dose level, the increased risk for a lymphoid malignancy after administration of isolated lymphocytes or mixed leukocytes labeled with 99mTc-HMPAO activities sufficient for scintigraphy can be regarded as small.

Invasiveness in in vitro and clinical evaluation of meningiomas.

Fragments of freshly isolated human meningiomas were cultured in vitro to form cell monolayers. These monolayers were confronted with embryonic chick heart fragments in vitro for 1, 2, 4, and 7 days. Microscopically, three different histological patterns were observed. Type I included necrotized meningial cells; type II presented surviving meningial cells; type III included meningial cells that had invaded the host tissue. The clinical analysis included the histopathological diagnosis, the macroscopic situation at surgical intervention, and the follow-up with or without recurrence. Correlation between these clinical parameters and the in vitro results demonstrated that type III confrontations correlated with macroscopic infiltration in the brain parenchyma and tumor recurrence. Invasiveness in vitro was seen in two anaplastic and two transitional meningiomas.

Invasiveness of human glioma cell lines in vitro: relation to tumorigenicity in athymic mice.

Five established cell lines derived from human anaplastic astrocytomas or glioblastoma multiforme were tested for invasiveness into precultured chick heart fragments in vitro. Four of the cell lines (U118 MG, D54 MG, U373 MG and A172) were strongly invasive into the heart tissue. A fifth cell line, U251 MG sp, which was only tumorigenic at doses of greater than 1 X 10(8) cells in athymic mice, was non-invasive in vitro. One line, A172, was invasive but not tumorigenic in athymic mice, although a related invasive subline, D54 MG, at later passage levels was tumorigenic even at low cell doses. Invasion of the glioma cells was characterized by progressive and irreversible replacement of the precultured chick heart tissue. Both by light and transmission electron microscopy, a similar pattern of invasion was observed as earlier found with experimental rat glioma cells in the same system. Some human cell lines established from human gliomas retain invasive properties after a prolonged culture period in vitro.

Invasion of rat neurogenic cell lines in embryonic chick heart fragments in vitro.

Invasive properties of 15 continuous neurogenic rat cell lines were investigated in vitro and were compared to their tumorigenicity in inbred BD IX rats. The cell lines were obtained by treating animals with a single transplacental dose of the carcinogen N-ethyl-N-nitrosourea (ENU) and 1) subsequently transferring brain cells into cell culture shortly after treatment or 2) explanting the resultant tumors from the offspring to monolayer cultures. In addition, one continuous nonneoplastic rat fibroblast line and three samples of untreated fetal rat brain cells were investigated. Cells from monolayer cultures were suspended and allowed to form aggregates for 24 hours on a gyratory shaker. The cell aggregates were then brought into contact with and allowed to attach to fragments from 9-days embryonic chick heart and were cultured on a gyratory shaker. All tumorigenic cell lines invaded the heart fragments, as characterized by progressive replacement of heart cells by invading cells. The heart tissue degenerated irreversibly. Nontumorigenic cell lines did not show invasiveness in vitro. Confrontation of cell aggregates and heart fragments in organotypic culture appeared to be a useful method to study directly the invasive properties of malignant transformants of neurogenic cells. The method might also permit prediction of tumorigenicity in the animal.