In vivo clonogenic tumor cell kinetics following 1,3-bis(2-chloroethyl)-1-nitrosourea brain tumor therapy.

An in vitro colony formation assay was modified to determine the effects of in vivo 1,3-bis(2-chloroethyl)-1-nitrosourea therapy on tumor cell kill and subsequent clonogenic cell kinetics. The measured surviving fraction must be multiplied by the relative total number of tumor cells for each posttreatment interval in order to eliminate inaccuracies caused by dead cell removal in vivo and the lysis of damaged cells by the disaggregation procedure. The assumptions, limitations, and applications of the technique are discussed. 1,3-Bis(2-chloroethyl)-1-nitrosourea doses of 0.25, 0.50, and 1.00 X dose lethal to 10% of animals resulted in approximately at 1-, 2-, and 3-log cell kill, respectively. Significant proliferation of surviving clonogenic cells was observed after a latency period of approximately 2 days, and the rate of tumor regrowth was dose dependent. The cell-doubling times following treatment with 0.25, 0.50, and 1.00 X doses lethal to 10% of animals were 15, 21, and 38 hr, respectively. The interval to complete repopulation of the clonogenic pool corresponds to the observed increase in animal life-span for the 2 larger doses and further validates the assay as a true measure of in vivo chemotherapeutic efficacy.

In vitro evaluation of in vivo brain tumor chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea.

An in vitro colony formation assay was used to determine the efficacy of in vitro therapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on a rat brain tumor. The fraction of clonogenic cells surviving in vivo therapy was determined by a comparison between the in vitro colony-forming capacity of cells derived from previously treated and untreated tumors. With this intracerebral solid tumor a direct correlation was found between the surviving fraction of cells and animal survival, implying that the in vitro assay system is a reliable test of therapeutic effect. The BCNU dose-response curve was exponential up to a dose of 0.75 times the LD10 dose with little additional cell kill noted at higher drug levels. This plateau does not appear to represent a resistant subpopulation of cells, since retreatment of tumors derived from cells surviving an LD10 dose were as sensitive to BCNU as those with no prior drug exposure. Instead, it may represent, at least in part, failure of the drug to reach and/or enter cells in all parts of solid tumors. On the average BCNU doses of 0.75 times the LD10 dose or greater resulted in slightly more than a 3-log cell kill and doubled the life-span for our tumor-bearing animals. The finding that an increase in animal life-span requires at least a 1-log tumor cell kill indicates that survival studies with intracranial tumor models may be insensitive to single courses of many chemotherapeutic agents with modest but significant antitumor activity.

Brain-tumor therapy. Quantitative analysis using a model system.

A recently developed colony-formation assay has been used to evaluate in vivo 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) therapy of a transplantable rat brain-tumor model. A comparison of the in vitro colony-forming capacity of treated and untreated tumor cells permits calculation of the fraction of clonogenic tumor cells surviving in vivo therapy; The plateau that we previously observed o the BCNU dose-response curve is not the result of repair of potentially lethal damage, since no change in the 0.1% of surviving clonogenic tumor cells occurs during the first 2 to 4 days after treatment. Although reanalysis of the dose-response curve indicates that sublethal damage exists, its repair is probably minimal. The most likely explanation for the observed limitation of the BCNU effect is the drug's failure to reach all clonogenic cells. A dose of BCNU that kills more than 99.9% of clonogenic tumor cells within 30 minutes of treatment results in only a 60% decrease in tumor weight by Day 14. This disparity is explained by retarded removal of dead cells, and, along with a previously determined 90% cell-kill threshold necessary to appreciate increased animal survival, demonstrates the inherent limitations of measurements of tumor size (including brain scans and clinical patient evaluations) in evaluating the efficacy of brain-tumor therapy. Following at LD10 dose of BCNU the surviving clonogenic tumor cells increase in number after latency period of 2 to 4 days; during regrowth the cell doubling time is 40 hours. Marked variability in tumor response and regrowth was noted. The determination of information regarding disturbed tumor cell kinetics and tumor heterogeneityis essential for the proper planning of combination chemotherapy and multimodality regimens.

The distribution of nuclear DNA from human brain-tumor cells.

Flow cytometry (FCM) is a technique that measures the quantity of DNA contained in individual nuclei and records a frequency distribution of the DNA content per nucleus in the sampled cell population. Nuclei from a variety of human brain-tumor types were isolated by means of tissue grinding, purified by centrifugation through 40% sucrose (15 minutes at 4000 rpm), fixed with 10% formalin, stained with acriflavin-Feulgen, and analyzed by FCM. Profiles of DNA distribution in histologically benign tumors, such as meningiomas, pituitary adenomas, neuroblastomas, and low-grade astrocytomas, revealed a large diploid population (2C) with a few nuclei in DNA synthesis, as well as a small premitotic population (G2 cells) that contains a 4C DNA complement. In contrast, malignant gliomas, including glioblastomas, consist of more cells in DNA synthesis; these tumor cells show a highly variable distribution of ploidy consisting not only of diploid, and/or aneuploid, but also of triploid, tetraploid, and possibly octaploid populations. Also, a large variability between different regions of each tumor was always observed. In contrast, metastatic brain tumors, despite the fact that they contain a considerable number of cells undergoing DNA synthesis, demonstrate little variability within each individual tumor. The ability to rapidly characterize the cell populations of human brain tumors with FCM may enhance the effectiveness of their clinical management.

Perturbed cell kinetics of 9L rat brain tumor cells following dianhydrogalactitol.

Kinetic changes induced by dianhydrogalactitol (DAG) in cultured 9L rat brain tumor cells were studied using conventional autoradiography and flow cytometry. Cell cycle parameters for the untreated cell line were: G1, 8.5 hours; S, 8.2 hours; G2, 3.2 hours; M, 0.5 hour; and cell cycle time, 19.5--20 hours. Treatment with 5 micrograms/ml of DAG, which caused 89.8% cell kill as measured by the colony-forming efficiency assay, resulted in the following effects: (a) undisturbed progression of G2 cells toward mitosis; (b) disturbed progression of the cells in the S--G2 boundary; (c) extreme prolongation of DNA synthesis time; and (d) temporary accumulation in G2 phase of cells that were in G1 or S phase at the time of treatment.

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

An 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 (10(3) to 10(5) 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 10(4) 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 10(4) 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 log(10) cell kill, and it should prove to be valuable in developing more effective regimens for the treatment of solid tumors in animals and man.

Clonogenicity of multiple populations of human glioma cells in vitro sorted by DNA content.

Malignant human gliomas have a highly variable distribution of cell nuclei, consisting of diploid and/or other populations in terms of nuclear DNA content. In order to study in vitro clonogenicity of each population, dissociated or cultured human glioma cells were stained with 20 microM/ml of Hoechst 33342 dye (which stains viable DNA with minimal cell kill), and were sorted sterile into separate populations, based on specific nuclear DNA content, for clonogenicity assay. The colony-forming efficiency (CFE) of tumor cells plated immediately after disaggregation of the biopsy specimens ranged from 0.0044 to 0.149%, and the CFEs increased dramatically with successive passages (to 5 to 40%). The CFEs of the individual populations sorted according to DNA content were similar within individual tumors. These results suggest not only that malignant gliomas are composed of multiple populations in terms of DNA content, but also that each of these populations contain clonogenic cells. The morphologic structure of cells within and among colonies did not appear to relate to DNA content.