Influence of Location within a Tumor on Cell Survival as Measured by a Clonogenic Assay1

In order to elucidate the influence of the location of a cell within a tumor on cell survival, 12 to 16 samples from an individual 9L s.c. tumor were assayed for colony formation. These samples (4 to 6 mg) were taken from untreated, heated, or irradiated tumors, and the variances of the mean relative colony-forming efficiency (RCFE) for individual tumors or groups of similarly treated tumors were compared. These com parisons allowed determination of the effect of location and treatment on the intratumor variability in cell survival. Three factors can influence variability of the RCFE's across tumors: (a) inherent differences in Clonogenic capacities; (b) unequal dose distribution of the treatment agent; and (c) different sen sitivities of cells to the treatment. Considerable variability was found in the cell survival of control tumors, and treatment of tumors in air-breathing or nitrogen-asphyxiated rats with Xrays did not change the magnitude of this variability. However, the variance of the mean RCFE of heated tumors was an order of magnitude greater than that observed in either control or irradiated tumors. Of the three possible factors that could influence cell survival within a tumor, all were shown to contrib ute to the variances of the mean RCFE measured in 9L s.c. tumors. However, our study demonstrated that the relative contribution of each of these factors to the variance will prob ably depend on the treatment agent(s). INTRODUCTION Analysis of many tumor parameters in the laboratory or clinic requires taking samples or biopsies from the tumor and then assuming that the values determined for that portion of the tumor are representative of the whole. Among the types of information normally accrued in this manner are: cell kinetic parameters; presence of biochemical markers; concentration and binding capacity of hormone receptors; and drug uptake and binding kinetics. Recently, the Division of Cancer Treat ment proposed using a human tumor colony-formation assay (2, 3, 6) as a primary screen for new chemotherapeutic agents. This assay involves dissociating biopsy samples from solid tumors into single cells, treating the cells with various chemo therapeutic agents, and then assaying for colony formation to determine the potential effectiveness of these drugs. However, the ultimate usefulness of this assay may rely on the assump1Supported by Grants CA-11198 and CA-11051 from the National Cancer Institute of the NIH and by the United States Department of Energy under Contract DE-AC02-76EV03490 with the Department of Radiation Biology and Biophysics (assigned University of Rochester Report 3490-1833). 2 Recipient of funds from the United States Department of Energy Laboratory Graduate Participants Program. Present address: Section of Radiation and Tumor Biology, Department of Radiology, University of Utah Medical Center, Salt Lake City, Utah 84132. To whom requests for reprints should be addressed. Received March 24, 1980; accepted November 25. 1980. tion that cells from various locations within the tumor have the same or a similar sensitivity to these agents. This assumption has not been systematically evaluated for agents whose dose distribution should be much more uniform than that expected for drugs. Tumor cells could vary in their response to a given treatment because of differences in: (a) genetic sensitivities (e.g., the presence of temperature-sensitive mutants when treating with heat); (b) physiological environments (e.g., areas with low oxygen concentrations can vary responses to radiation or alter cellular metabolic rates that effect drug metabolism); or (c) dose distribution of treatment agents (e.g., drug delivery or temperature gradients). These conditions could be very impor tant in determining the efficacy of a clinical treatment but would not necessarily affect the sensitivity of cells excised and then treated in vitro. This study was undertaken to determine the variability in cell survival as measured by a Clonogenic assay of single cells dissociated from small tumor pieces sampled from different locations within a tumor. The resulting intratumor and intertumor variability in cell survival was compared among tumors of various sizes that were either untreated or treated with heat or X-rays. MATERIALS AND METHODS Tumor Model. The 9L rat brain tumor used in these studies has been described previously (4, 5, 7, 8). The tumor was grown s.c. in the inguinal region of male Fischer 344 rats (Charles River Breeding Laboratories, Wilmington, Mass.) and was induced by injection of 1 x 106 9L/Ro exponential culture cells. The growth rate, morphological characteristics, as well as the procedures for the in vivo-in vitro colony-forming assay have been described previously in detail for the 9L s.c. tumor (7, 8, 10). Briefly, the tumor weight-doubling time was about 48 hr for the first 13 days (up to 1 g) and about 109 hr for the next 10 to 15 days (10). The tumors were well vascularized, had a relatively uniform morphology, and had little necrosis, even in tumors exceeding 1 g (1, 8). For the colony-forming assay, the tumors were excised, minced, and trypsinized into single cells, and then they were counted and plated at several dilutions into 60-mm Petri dishes containing 5 ml of Eagle's basal medium with 20% fetal calf serum (7). The cells were enumerated under phase-contrast microscopy to obtain the tumor cell concentration. The effects of the treatment and disaggregation procedure were monitored using trypan blue dye exclusion; of the untreated and X-irradiated cells, > 90% excluded the dye. With heat treatment, the number of trypan blue-excluding cells greatly decreased; therefore, the CFE3 3 The abbreviations used are: CFE, colony-forming efficiency; CY, cell yield; RCFE, relative colony-forming efficiency.