Response of 9 L Rat Brain Tumor Multicellular Spheroids to Single and Fractionated Doses of 1 , 3-Bis ( 2-chloroethyl )-1-nitrosourea 1

This study was designed to examine the relative effect of each of four fractions of 1,3-bis(2-chloroethyl)-1 -nitrosourea (BCNU) against 9L rat brain tumor multicellular spheroids and to compare the results of the cell survival and growth delay assays. Similar levels of cell kill resulted when BCNU was administered either as single fractions of 1.5, 3.0, 4.5, or 6.0 u.g/m\ for 1 hr or as one to four fractions of 1.5 ÃŸg/m\that were administered sequentially for 1 hr each. Survival was increased if the assay was delayed until 24 hr after drug treatment, which indicates that 9L cells in spheroids recover from BCNU-induced potentially lethal damage. When BCNU was administered in 1.5-Mg/ml fractions, plating efficiencies depended markedly on the interval between the fractions. The 12-hr protocol produced an overall higher cell kill. Fractionation schedules of 24 and 36 hr produced less cell kill than did the other schedules. Survival plateaued for the last three treatments with BCNU in the 36-hr schedule. Cells in S phase at the time of administration of the initial 1.5/Â¿g/ml fraction of BCNU moved into dand G2-M phases by 12 hr after treatment. For time periods longer than 12 hr, cells began to appear in the BCNU-resistant S phase. Thus, the movement of cells into the drug-sensitive and -resistant phases after the first fraction correlates well with the corresponding overall cytotoxic effect produced by treatment with the combined BCNU (1.5 Mg/ml) fractions. For a higher concentration (3.0 ng/m\ for 1 hr), maximum cell kill was reached within the 12to 18-hr interval, after which cell kill plateaued. Cells were not found in the S-phase fraction 12 to 36 hr after the first treatment with 3.0 ng/m\; maximum cell kill for the fractionated protocols resulted at these times. Therefore, BCNU, which is classified as a cell cycle-nonspecific drug, can induce a partial synchrony in 9L spheroid cells, which determines the overall cytotoxicity produced by fractionated BCNU proto cols. Although spheroids did not shrink during or after exposure to BCNU, growth was retarded by treatment with all doses and schedules. An optimum time point for growth delay measurement could not be determined from the data. However, correlations between cell survival and growth delay were obtained with arbitrarily chosen end point volumes of four and ten times the volume at the time of treatment. 1This research was supported by NIH Grants CA-31868, CA-13525, and CA19992 and the Andres Soriano Cancer Research Fund. 2Visiting scientist at the Brain Tumor Research Center. Permanent address: Department of Neurosurgery, Kumamoto University Medical School, Kumamoto, Japan 860. 3To whom requests for reprints should be addressed, at the Brain Tumor Research Center, 783 HSW, University of California, San Francisco, CA 94143. Received April 19, 1983; accepted November 3, 1983. INTRODUCTION The effects of drugs and/or radiation on tumor cells have been studied extensively in vivo and in vitro (6). Even though drugs are administered in fractionated doses in the clinical setting, most experimental studies have examined the effects of singlefraction treatment schedules. Exponentially growing monolayer cultures are not suitable for the study of multifraction protocols; because of confluence, cell proliferation ceases after a few days. Similarly, because environmental conditions contribute to a rather short lifetime in culture, cells in plateau phase are of little value for the study of extended protocols. Thus, most studies of multifraction protocols have been conducted with animal models; the end points of these studies include cell survival, tumor regrowth delay, and cure. However, extrapolation of results from animal studies to a clinical application is difficult because reasons for the success or failure of therapeutic protocols used against animal tumor models generally are not understood. Spheroids are well suited for the in vitro study of multifractional treatments. Although in some respects the 3-dimensional cellular structure of spheroids makes them more complex than monolayer cells in culture, they are less complex than animal tumor models, which are affected by host responses. Experimental end points for spheroid studies include cell survival and growth delay and, while it is not possible to "cure" a spheroid, complete cell kill in a spheroid is essentially equivalent to cure of a tumor in situ (4, 12). Thus, results obtained with spheroids can be com pared directly to results obtained with both in vitro and in vivo systems. We report here the results of treating 9L rat brain tumor multicellular spheroids with several concentrations of BCNU4 using a 4-fraction protocol. The relative cytotoxic effects of each of the fractions were compared, and the results obtained using the cell survival assay were compared with those obtained using the growth delay assay. Results of a detailed study of the synchrony effects caused by the fractionated treatments are discussed. MATERIALS AND METHODS Monolayer Culture. Monolayer 9L cells were grown in CMEM con sisting of Eagle's minimum essential medium supplemented with 10% newborn calf serum, nonessential amino acids, and gentamicin (50 ^g/ ml). Cells from exponentially growing monolayer cultures were used to initiate all spheroids. Spheroid Initiation and Culture. Optimization of various factors that affect spheroid initiation and culture has been reported (9). For these experiments, 9L cells were harvested from exponentially growing mon4The abbreviations used are: BCNU, 1,3-bis(2-chloroethyl)-1 -nitrosourea; CMEM, complete medium; [3H]dThd, tritiated thymidine; PE, plating efficiency.