Contributions of cell kill and posttreatment tumor growth rates to the repopulation of intracerebral 9L tumors after chemotherapy: An MRI study

Abstract

The drought of progress in clinical brain tumor therapy provides an impetus for developing new treatments as well as methods for testing therapeutics in animal models. The inability of traditional assays to simultaneously measure tumor size, location, growth kinetics, and cell kill achieved by a treatment complicates the interpretation of therapy experiments in animal models. To address these issues, tumor volume measurements obtained from serial magnetic resonance images were used to noninvasively estimate cell kill values in individual rats with intracerebral 9L tumors after treatment with 0.5, 1, or 2 ؋ LD 10 doses of 1,3-bis(2-chloroethyl)-1-nitrosourea. The calculated cell kill values were consistently lower than those reported using traditional assays. A dose-dependent increase in 9L tumor doubling time after treatment was observed that significantly contributed to the time required for surviving cells to repopulate the tumor mass. This study reveals that increases in animal survival are not exclusively attributable to the fraction of tumor cells killed but rather are a function of the cell kill and repopulation kinetics, both of which vary with treatment dose.Brain tumors occur frequently in the human population with approximately 35,000 new cases of primary adult central nervous system tumors diagnosed in the United States each year (1). The 1,500-2,000 brain tumors reported annually in children constitute the largest group of solid pediatric neoplasms (2). Despite the use of multimodality therapy, the management of brain tumors in adults and children remains unsatisfactory. In particular, the treatment of glioblastoma multiforme constitutes a major problem caused by the lack of therapeutic responses and a median survival time of only 1 year from the initial diagnosis (3).Promising agents identified through in vitro screening assays with brain tumor cell lines subsequently are evaluated in vivo by using animal brain tumor models (4-6). These studies frequently use rodent brain tumor models (7-10), particularly the rat 9L tumor (11-29). Animal survival, colony-forming efficiency (CFE) assays of cells disaggregated from solid tumors, and measurements of excised tumor weights have been used for two decades to quantitate the efficacy of various treatments on the orthotopic 9L brain tumor (11)(12)(13)(14)(15)(16)(17)(18)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Numerous in vitro and in vivo studies have shown that 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is an effective treatment for the 9L brain tumor (11-15, 17, 18, 20-23, 25-29); in fact, a single bolus of BCNU achieved a 3-4 log cell kill in orthotopic tumors, resulting in a 97% increase in life span (13).BCNU is a mainstay of brain tumor chemotherapy, but the clinical outcome of patients treated solely with BCNU has not proven as efficacious as would be predicted by the responses observed in the 9L tumor model (30-33).In the present study, MRI was used for noninvasive estimation of tumor cell kill in individual animals with orthotopic 9L brain...

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“…344 rats, weighing 125-150 g, according to a previously described procedure [17]. Briefly, rat 9L-glioma cells (Brain Tumor Research Center, University of California, San Francisco) were cultured at 37°C, in a humidified atmosphere of 5% CO 2 , in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin and 0.29 mg of L-glutamine.…”

“…Thirteen axial sections of the rat brain were acquired with a T 2 -weighted fast spin echo sequence using the following parameters: repetition time (TR) = 4 s, echo time (TE) = 60 ms, field of view = 30 × 30 over 128 × 128 matrix, slice thickness = 1mm, slice separation = 1.5 mm, four signal averages per phase encoding step. Tumor volumes were calculated from the collected images as previously described [17].…”

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“…The enhancing tumor volume was delineated based on pre-and post-contrast T1-weighted images, and a consideration of the total tumor volume, based on the inherently co-registered T2-weighted images. Tumor and enhancing tissue volumes were derived from these multi-slice MR data sets over time for each individual animal as previously described (47). At 7.5 weeks of age, half of the mice were sacrificed for histology, with the remaining mice continuing on study for survival analysis.…”

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