Comparative Rates of Dead Tumor Cell Removal From Brain, Muscle, Subcu- taneous Tissue, and Peritoneal Cavity 1, 2 A. R. V. Kumar, T. Hoshino, K. T. Wheeler,3 M. Barker,' and C. B. Wilson 5,6 SUMMARY-Two methods were used to compare the rates of dead cell removal from pieces of tumor implanted in brain, muscle, subcutaneous (sc) tissue, and peritoneal cavity of rats. The weight of implanted tumor pieces, rendered nonviable either by lethal irradiation or by several freeze·thaw cycles, was followed as a function of post· implantation time. A large initial weight increase occurred in tumors implanted in sc tissue and brain, but not in muscle. The subsequent weight reduction of implanted tumors indicated that the rate of dead tumor·cell removal was greatest in muscle, intermediate in sc tissue, and least in brain. The loss of 125 1 label from dead tumor pre· labeled with 125 2' -deoxy·5·iodouridine was followed during the postimplantation period by counting either the whole animal or the isolated DNA from excised tumor masses. Loss of the 125 1 label from tumor implants in situ and from extracted DNA failed to correlate with the removal of dead cells.-J Natl Cancer Inst 52: 1751-1755, 1974. A TUMOR'S growth rate is determined by its cell- cycle time, growth fraction, and extent of cell loss (1-3). Spontaneous cell loss represents a significant factor in the life history of many human and animal tumors (3-5). Furthermore, nonneoplastic elements within a tumor possess kinetic characteristics that must be considered in any analysis of volumetric changes. Beyond the period of log phase growth, a solid tumor contains the following neoplastic and nonneoplastic components: tumor cells-I) living cells capable of unlimited cell division, 2) living cells without capacity to divide, 3) dead cells; host cells-I) monocytes, 2) phagocytes, 3) fibroblasts; other components- 1) blood vessels, 2) stroma (noncellular components), 3) hemorrhage, 4) acellular debris. Because necrotic tissue occupies a variable but significant volume in most malignant brain tumors, the rate of dead cell removal becomes an important factor in defining their kinetic behavior, particularly after treatment·induced cell death, e.g., chemo- therapy. The present study was designed to develop a method for the investigation of the kinetics of dead cell removal in general and to compare the efficiencies of dead tumor-cell removal from brain, muscle, subcutaneous (sc) tissue, and peritoneal cavity. Prusoff (6, 7) synthesized 131 2' -deoxy-5-iodouridine 131(IUDR) in 1959 and later demonstrated its incor- poration into the DNA of mouse Ehrlich ascites cells. The stability of 125IUDR as a radiotracer for studying DNA metabolism (i.e., the label is not removed from intact DNA in living cells) was shown by Commerford (8). Hofer et al. (9) and Dethlefsen (10) reported that only a small percentage of the incorporated IUDR is reutilized if the diet is supplemented with sodium iodide. Since the low energy X-ray from 125 1 can be detected with externally positioned gamma detectors, test animals need not be killed to study cell removal kinetics. Several investigators (8, 11-13) suggested that 125IUDR may be the compound of choice for following cell loss in vivo. However, results obtained here indicate that the in vivo loss of 126 1 from labeled dead tumor implants does not predict the rate of cell loss from the tissues in question. MATERIALS AND METHODS Weight reduction oj dead tumor.-Cells in flank glioma tumors were killed by 4 cycles of freezing (-80 0 C) and thawing (room temperature). The tumor was cut into 3-4 mm 3 pieces and weighed; the pieces were implanted into brain (20 rats), muscle (20 rats), and sc tissue (20 rats). Intracerebral implan- tation was performed with microneurosurgical tech- niques in rats anesthetized intraperitoneally (ip) with pentobarbital. A small craniectomy and partial removal of the frontal lobe were performed before the labeled tumor mass was implanted. Implantation of tumor into muscle and sc tissue was accomplished by simple surgical methods. Two or 3 rats in each group were killed at 2, 4, 7, 10, 14, 16, and 18 days after implantation. The implanted tumor, if any, was removed, weighed, and fixed in 10% formalin for histologic examination. Labeling, cell killing, and implantation of tumor.- Tumors were grown in the flanks of male Fischer 344 rats by the injection of 10 6 syngeneic rat glioma cells. The characteristics of this tumor were reported (14). When the tumor reached 8-10 mm in diameter, the tumor was labeled with four 250 p.Ci ip injections of 125IUDR at 6-hour intervals. One to 2 hours after the last dose, the tumor cells were killed by irradiation of the tumor in vivo with 5 krads of X-rays (300 kV, 20 rnA, HVL 1.62 mm Cu) or by at least 4 cycles of freezing (-80 0 C) and thawing (room temperature). The tumor was cut into 2-3 mm 3 pieces (30-100 mg) and then implanted into the white matter of brain (15 rats), sc tissue (15 rats), muscle (15 rats), and peritoneal cavity (15 rats). 1 Received October 23, 1973; accepted February 25, 1974. 2 Supported in part by Public Health Service research grant CA13525 from the National Cancer Institute, a gift from the Phi Beta Psi sorority, a gift from the Joe Gheen Medical Foundation, and by the U. S. Atomic Energy Commission. 3 Also Laboratory of Radiobiology, University of California. 4 Please send all communications to M. Barker. 5 Naffziger Laboratories for Neurosurgical Research and the Department of Neurological Surgery, University of Cali- fornia, San Francisco, Calif. 94143. 6 The tumor used was originally provided by William H. Sweet, Paul T. Kornblith, Janette R. Messer, and Beverly O. Whitman of the Massachusetts General Hospital, Boston, Mass. 02114. The technical assistance of Kathy Knebel and Maria Misiaszek is greatly appreciated. JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 52, NO.6, JUNE 1974 1751 Downloaded from https://academic.oup.com/jnci/article-abstract/52/6/1751/923424 by guest on 01 March 2019