Rates of Cell Division of Transplantable Malignant Rat Tumors*
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Rates of Cell Division of Transplantable Malignant Rat Tumors* FELIXD. BERTALANFFYANDCHOSENLAU (Department of Anatomy, Faculties of Medicine and Dentistry, Unirersity of Manitoba, Winnipeg, Manitoba, Canada) SUMMARY The mitotic rates of transplantable Walker carcinosarcoma 256 and fibrosarcoma 1F16F were investigated in rats by the colchicine method. The mitotic rates of these tumors were apparently not influenced by the time of day. In female rats the estrous cycle did not seem to have appreciable effects on the mitotic rates of fibrosarcoma. During the period of active growth of the tumors a constant increase in the number of cells occurred each day until the onset of necrosis. During the 5th-10th day after transplantation about 60 per cent of the cells divided daily in Walker carcinosarcoma. During the growth period of the fibrosarcoma—i.e., from the 15th to the 32d day after transplantation—about 40 per cent of newly formed cells were daily added to this tumor. These figures imply that the cell population of Walker tumor doubles about every 1.7 days, of the fibrosarcoma every 2.5 days. The mitotic rates of these malig nant tumors exceed those of most normal tissues and are surpassed only by those of the epithelium (crypts) in the small intestine. Tumor growth has been quantitatively esti plicable only to solid tumors. Disadvantages were mated by a variety of methods. Radioactive trac that malignant tumors rarely are exactly spheri ers, such as tritiated thymidine (e.g., 11), spectro- cal, cylindrical, or conical, as is requisite for the photometric determination (e.g., 22, 23) of DNA application of the above formulas. Moreover, for synthesis and content of malignant tissues have example, the Walker carcinosarcoma 256 is not been employed. Other methods entailed measure necessarily solid throughout. When applying the ment of the size of tumors. Various formulas have formulas, particularly at later stages after trans been proposed to calculate, particularly, the vol plantation, necrotic debris and fluid in the center ume of tumors. Gaylord and Clowes (13) calcu of the tumors, the result of degenerative processes, lated the volume of mass of tumors by the formula would be included as viable parts of the tumor. V = (7T/6)¿i¿22;d\and ¿2represent two diameters A more direct method for the determination of of the tumor. Woglom (21) used the formula V = tumor growth is thus desirable. The colchicine (4/3) irr3; r represents the average radius. Cham technic enables one to overcome to a large extent bers and Scott (9) considered the volume to be a the above-mentioned difficulties. Size and shape of function of (\X06)3; a and b represent two diam the tumor, necrosis, and other factors do not eters of the tumor. Schrek (18) estimated the vol hamper measurement of the mitotic rate by the ume of Walker tumors with the formula V = colchicine technic. Such factors as cell debris and 0.5236d3; d represents the geometric mean of three necrotic fluid do not present difficulties. Moreover, diameters of the tumor (the cube root of product growth of métastases, as well as the mitotic rates of the three dimensions). Tumor growth has been of any type of animal tumor can be determined. estimated by graphically plotting the calculated Colchicine, when injected into rats in a dose of volume against time. The slope of the graph would 0.10 mg/100 gm of body weight, arrests in meta- represent the rate of growth of the particular tu phase those cells that have entered division during mor. the time interval between injection and sacrifice Volume measurements of tumor growth are ap- of the animal (12). Enumeration of resting cells and "colchicine metaphases" permits calculation of * This work was supported by a research grant from the the percentage of cells that divide in a tissue dur National Cancer Institute of Canada, Toronto. ing a certain period of time (15, 16). It is thus Received for publication December 11, 1961. possible to estimate the percentage increase of 627 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1962 American Association for Cancer Research. 628 Cancer Research Vol. 22, June 1962 newly formed cells in a tumor during 1 day (the palpable 15 days after transplantation. Therefore, daily mitotic rate) and, further, the time required beginning with the 15th day, four animals were for the addition of 100 per cent of tumor cells. given injections of colchicine at 10:00 A.M. and In some animal tissues, the mitotic activity ex sacrificed 6 hours after injection. The colchicine hibits appreciable diurnal fluctuations during the experiments were carried out on the 15th, 18th, 24-hour period, whereas other tissues do not dis 22d, 26th, 28th, and 32d day after transplantation. play any daily rhythm. It has been reported (8,14) Beyond this the tumors had become extensively that experimentally produced epidermoid carci necrotic, and most became unsuitable for mitotic nomas in mice did not display diurnal variations counts. To ascertain whether a diurnal pattern of of mitotic rate, the latter remaining at an almost mitotic rate was apparent in this tumor, sixteen constant level throughout day and night. A similar animals were given injections of colchicine at 4 finding was made on human carcinoma of the large different times of the day (as above), 32 days after intestine (10). transplantation. In the present investigation the mitotic rates of Following sacrifice, tissue samples from non- the Walker 256 carcinosarcoma and fibrosarcoma necrotic areas were collected from each tumor mass 1F16F in the rat were determined, from the time and immediately fixed in sublimate formalin. Par sufficient tissues could be obtained after trans affin sections, cut at 7 p.,were stained with hema- plantation until the onset of extensive necrosis, toxylin and eosin. With the aid of a hand tal which automatically terminated the experiment, ly counter, more than 2000 resting nuclei were often by killing the animals. Moreover, we investi counted (with medium-high magnifications) in tu gated whether these tumors underwent a daily mor sections of each animal; the numbers of col rhythm of mitotic activity or whether they be chicine metaphases that occurred in the counted haved similarly in this regard to the above-men fields were recorded separately. The percentage of tioned epidermoid and intestinal carcinomas. cells that divided during the 6-hour period of col chicine action was calculated for each animal. In MATERIALS AND METHODS the two 24-hour experiments, each composed of Walker carcinosarcoma.—Adult,male albino rats four groups of four rats (and each group covering of the Sprague-Dawley strain (Holtzmann Rat a different 6-hour period of the day), summation of Co., Madison, Wis.), ranging from 7 to 12 months, the four 6-hour percentages yielded the percentage were given transplants of Walker rat tumor tissue of cells that divided during 1 day—i.e., the daily (Carcinosarcoma 256). The tumor tissue was finely mitotic rate of the particular tumor. Knowing the minced and a saline suspension prepared. Of this percentages of cells that divided in the tissues dur tumor cell suspension, 0.25 cc. was injected into ing 1 day, one can readily calculate the number of both thighs (hindlegs) of each rat. Each day, from days required for 100 per cent addition of tumor the 5th to the 10th day after the tumor transplan cells. tation, four rats were given injections at 10:00 A.M. of 0.10 mg colchicine/100 gm of body weight; they RESULTS were sacrificed exactly 6 hours later. With Walker carcinosarcoma no demonstrable To ascertain whether diurnal fluctuations of tumor mass could be observed prior to the 5th day mitotic rate occurred in the Walker tumor, sixteen after transplantation; thus, not sufficient tissue rats, on the 7th day after transplantation, were di was available for mitotic counts at earlier stages. vided into four groups of four animals each. The With the tumor suspension used, few of the ani animals of each of these groups were given injec mals survived longer than 14 days. Actively grow tions of colchicine at a different time of the day— ing cancerous tissue was present throughout the i.e., at 10:00 A.M.,4:00 P.M., 10:00 P.M.,and 4:00 tumor mass up to the 7th day after transplanta A.M.They were sacrificed 6 hours after injection. tion. Subsequently, the central portion of the tu Fibrosarcoma.—Adult female rats, of similar mor underwent progressive necrosis, became lique strain, ranging from 7 to 12 months, were trans fied, and contained degenerated cell debris. After planted with fibrosarcoma 1F16F—54FM tumor the 9th day, most of the tumor consisted of necrot (20)—tissue. Non-necrotic fibrosarcoma tissue was ic debris, and the growing tumor tissue was con removed from animals and cut in saline into small fined to a peripheral zone. In deeper regions, the pieces, about 2 mm. in diameter. These were in mitotic rates varied greatly, depending on the de serted into anesthetized rats through a small inci gree of necrosis in the samples of tissue. Therefore, sion over the xiphoid process beneath the epider determination of the mitotic rate was confined to mis into the subcutaneous tissue. The incision was the periphery of the tumor, which was best pre subsequently closed with a metal clip; the latter served. Subsequent to the 10th day after trans was removed after a week. A small tumor mass was plantation, mitotic counts became impossible be- Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1962 American Association for Cancer Research. BERTALANFFYANDLAU—CellDivision in Rat Tumors cause enough well preserved tumor specimens could group for each 6-hour period.