Marvin Barker

The 9L rat brain tumor: Description and application of an animal model

SummaryAnimal models allow determination of tumor response to anticancer agents under various experimental conditions. The chemically induced 9L rat brain tumor has been developed as both in vivo and in vitro models. Animal survival, clonogenic cell survival, and tumor growth delay provide means to measure the effectiveness of treatment modalities in this tumor model. Monolayer cultures, multicellular spheroid cultures, brain tumors, and flank tumors have been used to study the influence of different biological entities of the 9L model on the response to treatment with radiation and/or BCNU (1,3- bis (2-chloroethyl)-1-nitrosourea).ZusammenfassungDie Behandlung von Tumoren unterliegt den Einflüssen verschiedener Umgebungsfaktoren, die an Tiertumormodellen erforscht werden konnten. Für den chemisch induzierten 9L Hirntumor der Ratte wurden in vivo- and in vitro-Systeme entwickelt. Überlebensraten von Tieren oder clonogenen Zellen und Wachstumsverzögerung von Tumoren können gemessen werden, um die Wirksamkeit verschiedener Tumorbehandlungen zu bestimmen. Monolayer- und Spheroidkulturen sowie intracerebral oder subcutan wachsende Tumoren wurden verwendet, um den Einfluß unterschiedlicher Tumorgestalt auf die Wirkung von Strahlen und/oder BCNU (1,3- bis (2-chloroethyl)-1-nitrosourea) bei 9L Zellen zu demonstrieren.

BCNU and x-ray therapy of intracerebral 9L rat tumors

Abstract Combination therapy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and X-rays was applied to the 9L rat brain tumor model. BCNU alone (13.3 mg/kg) and X-rays alone (2,000 rad/whole brain) were given on day 16 postimplant. The two therapies were combined in three schedules with BCNU given 6 hours before, 6 hours after, and immediately preceding X-ray treatment. While either modality alone significantly increased survival, the combined therapies produced increased life spans (ILS) of 235 to 430%. The combined therapies also resulted in cure rates as high as 60%. Some toxicity was encountered as evidenced by interstitial pulmonary fibrosis when “cured” and treated non-tumor bearing animals were autopsied on day 122 or day 125.

Development of an in vitro colony formation assay for the evaluation of in vivo chemotherapy of a rat brain tumor

SummaryAn in vitro colony formation assay for the evaluation of in vivo brain tumor therapy has been developed. When plated, disaggregated cells derived from solid tumors proliferated to form relatively homogeneous colonies after a latency period of 2 to 6 days. Increasing concentrations of fetal calf serum enhanced colony-forming efficiency (CFE) with a plateau between 7 and 16%. Supplementation with either irradiated feeder cells (103 to 105 cells per dish), or medium conditioned by 1 to 3 days of in vitro incubation with the same cell line, doubled the CFE. The density of tumor cells (untreated or previously treated with chemotherapeutic agents) did not affect the CFE when a minimum of 104 total cells (tumor plus feeder) were plated. Therefore, in this system the optimal experimental conditions for evaluating chemotherapy and radiotherapy require incubation of disaggregated tumor cells for 12 days in medium containing 10% of fetal calf serum and enough feeder cells to provide a minimum of 104 cells per dish. The CFE for untreated tumors was 18±10% (±S.D.), demonstrating that there is significant biological variation.The assay appeared sensitive, with reproducible results, when applied to individual chemically treated tumors. An estimate of the percentage of clonogenic cells affected by in vivo chemotherapy may be obtained by comparing the CFE of cells from treated and untreated tumors. This assay can measure up to a 5 log10 cell kill, and it should prove to be valuable in developing more effective regimens for the treatment of solid tumors in animals and man.

Chromosome analysis of glioblastoma multiforme

SUMMARYEleven glioblastomas were submitted for chromosome analysis. Satisfactory cells were obtained from 3 as fresh tumor tissue and from the remainder after short periods in cell cultures. The predominant pattern was a near-diploid karyotype, with most frequent deviations occurring in Group 6-12. A review of earlier reports and results of the present study indicate the frequent occurrence of the above pattern in glioblastomas, with some tumors also containing an additional population of near-tetraploid cells. One established glioblastoma cell line had a hypotriploid karyotype and 4 marker chromosomes.

Effect of dibutyryl cAMP on cell cycle progression of rat brain tumor cells in vitro.

SummaryAutoradiographic and flow microfluorometry analyses have been applied to a study of perturbed cell kinetics in 9L rat brain tumor cells treated with dibutyryl cyclic AMP and theophylline alone and in combination in vitro. At a concentration of 1 mM each, cell growth ceased shortly after the administration of these drugs. The results indicate that cells in S and G2 phase at the time of drug administration can undergo mitosis even though a considerable prolongation of G2 phase was apparent. However, cells in G1 at the time of drug administration were arrested in that phase whereas those cells in S or G2 were able to complete one mitosis before becoming arrested in the G1 phase. This blocking effect was reversible, and cells resumed proliferation at a normal rate shortly after the removal of these drugs.

Subhypothalamic grafts of human pituitary adenomas in total-body irradiated rats

SummaryHuman pituitary adenomas proliferate neither in cell culture nor in athymic “nude” mice. We propose that one or several of the humoral factors necessary for the growth of pituitary adenomas is missing in these experimental environments. The purpose of our experiments was to examine the possible influence of the hypothalamus in supporting cellular proliferation, and thus adenoma growth. Fragments from four human pituitary adenomas (three pituitary prolactinomas; one ACTH-secreting adenoma) were transplanted into the pituitary fossa of total-body irradiated, hypophysectomized rats. The rats were killed after two weeks and perfused with a mixture of formalin and India ink. Histologic examination of serial sagittal sections through the pituitary fossa and the adjacent brain showed: vascularization of the grafts from the pituitary stalk and from the scar tissue in the sphenoid bone; survival of some adenomas; and numerous mitoses in an ACTH-secreting specimen obtained from a patient who had Cushings disease. We conclude from these experiments that as yet unidentified hypothalamic factors are essential for the growth of certain types of pituitary adenomas.

The kinetics of cultured human glioma cells

SummaryThe kinetics of monolayer cell cultures, derived from brain tumors which had been labeled with3H-thymidine just prior to surgical removal, have been investigated. Analysis of autoradiographs indicates that: 1. the distribution of cell types in primary cultures is similar to that of the viable areas of the parent tumorin vivo; 2. the first week of culture constitutes a lag phase; and 3. vigorous proliferation commences during the second week of culture. Double labeling of the tritiated cultures with14C-thymidine indicates that the pulse labeling index (PLI)* of primary cultures during the first week is lower than that of the parent tumor and that the PLI of the culture increases during the second week. In contrast, we found that long-term established cultures in our laboratory have a high PLI during the first few days after passage, quickly reach a maximum, and then drop precipitously 1 week after passage. These latter variations in PLI correspond to the following growth pattern: 1. exponential growth; 2. a stationary phase due to medium exhaustion; or 3. a plateau due to overcrowding. There are indications that some cell types, which have the capacity to divide in the parent tumor, lose this ability after transfer to tissue culture.

Cerebrospinal fluid as a culture medium for human brain tumors

Two OBSERVATIONS SUGGEST viability of tumor cells in cerebrospinal fluid (CSF). First, cytological examination of CSF from patients harboring tumors of the central nervous system has revealed tumor cells which appear to be viable by morphological criteria. Second, certain tumors, notably medulloblastomas, metastasize by way of CSF to form implants along the subarachnoid space and within the cerebral ventricles. Unable to find previous reference to the behavior of tumor cells in CSF, we undertook a study of CSF as a nutrient medium for cell cultures of primary brain tumors. Using conventional tissue culture methods, we have demonstrated the capacity of CSF to support growth and multiplication of tumor cells. Our experience forms the substance of the present report.

Experimental basis for increasing the therapeutic index ofcis-diamminedicarboxylatocyclobutaneplatinum(II) in brain tumor therapy by a high-zinc diet

SummaryMetallothionein (MT), a ubiquitous intracellular protein, confers resistance to the toxic effects of platinum compound. Since a high-zinc diet has been shown to induce MT synthesis in extracerebral tissues but not in brain, we investigated whether it could provide an experimental basis for decreasing the hematotoxicity of carboplatin without impairing its activity against brain tumors. After 2 weeks on either a high-zinc diet or a control diet (zinc content, 180 vs 10 ppm), mice and rats received various doses of carboplatin or Hanks balanced salt solution by i.p. injection. The hematotoxicity of carboplatin was evaluated with an assay of colony-forming units of granulocytes and mononuclear cells in mice. The high-zinc diet enabled a 50% increase in the carboplatin dose without increasing hematotoxicity. The antitumor activity was evaluated with an assay of the colony-forming efficiency of gliosarcoma cells from 9L brain tumors in rats. The high-zinc diet did not alter the efficacy of carboplatin against this brain tumor. Northern blot analysis confirmed that the high-zinc diet induced MT mRNA in the kidney but not in the brain of mice and rats: it also showed MT mRNA induction in bone marrow cells of mice but not in rat 9L brain tumors. These results suggest that increasing the dietary intake of zinc might increase the therapeutic index of carboplatin in the treatment of brain tumors.