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The relative resistance of malignant glioma to chemotherapy makes the identification of new cytotoxic drugs critically important. The use of short-term cultures derived from these tumors to screen drugs at doses that can be attained within human intracranial tumors provides a model system that should be capable of identifying effective drugs suitable for clinical evaluation. The sensitivity of a panel of short-term cultures derived from 22 malignant astrocytoma and four malignant oligodendroglioma was assessed to aziridinylbenzoquinone (AZQ), etoposide and doxorubicin (DOX) using a [(35)S] methione uptake assay. The ID(50) of each culture was compared to the levels of drug which could be achieved in the tumor using standard doses. There was marked heterogeneity between cultures in response to each drug. Whilst there was no evidence that cultures derived from grade III astrocytoma were more sensitive to any of the drugs than cultures derived from grade IV astrocytoma, cultures derived from oligodendroglioma tended to be more sensitive to the alkylating agent AZQ, but not to either of the other drugs. The sensitivity of these short-term cultures at concentrations that can be achieved in situ corresponded well with the clinical efficacy of AZQ and etoposide. Although DOX appeared to be toxic to human gliomas cells in vitro, its limited penetration into the intact brain would seem to preclude its use i.v., but it is likely to be effective if local drug delivery techniques could be employed. The study suggests that short-term cultures derived from malignant glioma should be used to screen investigational agents for potential clinical efficacy.

We describe a simple method for detecting [35S]methionine-labeled protein in fixed human astrocytoma cells grown in 96-well microtitration plates using a modified scintillation autofluorographic method. Following isotopic labeling, cells are fixed in situ and a solution of salicylic acid in methanol is dried onto the cell layer. The fluorographic image is detected using blue-sensitive X-ray film attached to the base of the plate which, following development, can be quantitated using a scanning densitometer. The relationship between cell number and optical density is linear, and there is a close correlation between the dose-response curves generated by this method and alternative isotopic detection methods and cell counting. This assay provides a suitable alternative to the use of potentially toxic scintillation fluids based on organic solvents like toluene or xylene in chemosensitivity testing of human brain tumors.

The activity of temozolomide, which has shown clinical activity against malignant glioma, has been assessed in vitro against short-term cultures derived from these tumors using an intermediate duration microtitration assay with MTT reduction as the end-point This assay has previously been shown to correlate closely with a monolayer clonogenic assay. Sensitivity was assessed in 15 short-term cultures (passage levels 3-9) derived from WHO grade III and IV astrocytomas. These cultures had a median ID50 value of 258 microM for temozolomide and 16.13 microM for CCNU. Maximum serum concentrations of temozolomide are of the order of 75 microM but only three of 15 (20%) cultures had ID50s below this value. Fourteen of 15 (93%) cultures displayed cross-resistance between temozolomide and CCNU, although one line which was extremely resistant to CCNU retained sensitivity to temozolomide. Comparative studies of published clonogenic survival curves indicate that the short-term glioma cell lines used in this study have similar sensitivities to established glioma cell lines, whilst colon carcinoma cell lines and bladder carcinoma are often more resistant to these drugs. Cell lines from testicular teratoma cell lines may show exquisite sensitivity to temozolomide and this level of sensitivity is seen only occasionally in short-term cultures derived from malignant glioma.

Because of the methodological difficulties associated with the MTT assay in screening short-term cultures derived from human malignant glioma, a chemosensitivity assay based on the protein staining using sulforhodamine B (SRB) has been optimized for use with these cells. SRB at a fixed dye concentration achieved maximal staining density at 20 min for most cell lines and this intensity was not further increased by using dye concentrations above 0.2%. A delay in staining after fixation did not significantly decrease staining intensity, but delay in dye extraction after fixation and staining did. There was an excellent quantitative and qualitative linear relationship between cell number determined by either the SRB assay or by cell counting, but not with the MTT assay which consistently underestimated the number of cells in assay plates. The MTT assay appeared to be incapable of detecting less than about 150 cells/well, while these small numbers of cell were readily detectable by either cell counting or SRB staining. There was a close correlation between chemosensitivity values derived from the MTT and SRB assays for procarbazine, CCNU and vincristine when the endpoint is taken as either the ID25, ID50 or ID75. The results indicate that the SRB is capable of producing broadly similar results to the MTT assay, but is more sensitive in the detection of small numbers of cells with a linear relationship between cell number and SRB staining intensity over a wide range of cell numbers. It is capable of producing data from short-term cultures from malignant glioma and offers technical advantages over the MTT assay in that plates may safely be stored at certain points during the assay without the need for immediate processing. The SRB assay provides a useful alternative to the MTT assay for determining the sensitivity of short-term cultures of human glioma to cytotoxic drugs.