Kinetics of Cell Proliferation of an Experimental Tumor
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(CANCER RESEARCH 27 Part 1, 1122-1131,June 1967] Kinetics of Cell Proliferation of an Experimental Tumor EMILIA FRINDEL, EDMOND P. MALAISE, EDWARD ALPEN', AND MAURICE TUBIANA Institut Gustave-Roussy, Villejuif, Seine, France SUMMARY logie character of the tumor. For this reason, it seemed to us that this tumor would lend itself ]>articularly well to studies of evalua The cellular proliferation kinetics of an exjxrimental fibro tions of the kinetics of proliferation of individual cells during sarcoma in C3H mice have been studied in vitro and in vivo at tumoral growth. In addition, this cell can be cultivated in vitro, various stages in tumor growth. and under these conditions, one also sees that the growth rate The duration of the cell cycle measured in vitro is the same as or to be more explicit, the rate of change of the cell number has that measured in vivo and does not change when the increase in an evolution analogous to those of the solid tumor. The number cell number, at first exponential, slows progressively. The slow of cells at first increases very rapidly, there is then a progressive ing down of the growth rate and the plateau in vitro are explained slowing in rate, and finally a plateau in cell number is reached. It mainly by a reduction in the proportion of cells engaged in the ap]>eared to us that a comparative study of the kinetics of pro cell cycle and by increasing cell death. liferation in vitro would be very interesting and might provide In vivo, the growth rate is at first rapid and then slows pro further insight into the in vivo process. gressively. The duration of the cell cycle is similar in all phases of tumor growth. A diminution both of the number of labeled MATERIALS AND METHODS cells after multiple injections of tritiated thymidine and of the growth fraction is seen as the growth rate slows. It is probable In Vi vo that in this case also increasing cell death contributes to the slowing of tumor growth. Autoradiographs in large tumors Cells. NCTC clone 2472 is cultivated in Medium 109 (7, 13) after multiple injections show considerable heterogeneity in with 10% added horse serum. The cells injected into the mice labeling from one region of the tumor to another. are obtained from a culture in exponential growth. The medium is renewed the day before. The cells are put into suspension using trypsin (1:300) solution ata concentration of 0.05f'.¿.After INTRODUCTION centrifuging, they are resusjiended in Medium 109. The final The jattern of growth of experimental tumors has been the cell concentration is 7,500,000 cells i>er ml. Into the flanks of object of recent general reviews (5, 6, 10) in which it is indicated C3H male mice aged 2 to 3 months, 750,000 cells (0.1 ml) are that, in solid tumor as well in neoplastic ascites tumors, the in injected subcutaneously. The pre|iaration and injection of the crease in the cell number, which is at first quite rapid, progres cells is completed within half an hour. sively decreases. Estimation of Tumor Volume. Two hundred mice aged Several paj)ers recently reviewed by Baserga (1) and 2 to 3 months were used to study the growth curve of the tumor. Mendelsohn (12) have been devoted to the kinetics of cell pro Measurements were done daily after the 6th day following the liferation in s|x>ntaneous and induced tumors. However, to our injection of tumor cells using a caliper. Two dimensions of the knowledge, no authors have studied the kinetics of cellular tumor were noted: the larges and smallest diameter. Usually, growth in the same type of solid tumors at different stages of they are at right angles; tumor thickness was not measured. their growth in order to analyze the causes of the decrease in From these two diameters (D and d) and taking into account growth rate which has been observed. It is possible to envisage the double thickness of the skin overlying the tumor (2 x 0.5 mm), several reasons for the observed decrease in growth rate, for the volume of the tumor (in cu mm) is calculated from the fol example, either a decrease in the growth fraction or an increase lowing formula: in the length of the cellular cycle. (D + d - 1) We have been studying for several years a fibrosarcoma of the 4/3- C3H mouse in which the growth rate is extremely reproducible from one animal to the next when one injects into a recipient the The volume which is obtained using this formula is larger than same number of cells coming from the same lot of cells cultivated the true volume. Tumor mass and apparent volume have been in vitro (8, 9). During the course of the first 20 days or so after compared in 100 animals sacrificed at variable times during the implantation, the pattern of growth is at first rapid and then evolution of the tumor. The results show that for all tumor sizes slows considerably without any significant change in the histo the ratio, apparent volume/tumor mass, varies little and equals, on average, 1.75. 1Present address: U. S. Naval Research Laboratory, San Fran Cell Cycle. The cell cycle is studied by the method of labeled cisco, California. mitoses. At 3 days, 7 days, and 20 days after inoculation of the Received September 19, 1966; accepted February 16, 1967. NCTC cells, 60 mice per group received 50 microcuries of thyini- 1122 CANCER RESEARCH VOL. 27 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1967 American Association for Cancer Research. Kinetics of Cell Proliferation of Experimental Tumor dine-3H intraperitoneally in a single injection and 4 mice of each group were sacrificed by cervical dislocation at various times, from 15 minutes to 40 hours after injection of the DNA precursor. The tumors were dissected and fixed in Carnoy's fixative. Sections 4 microns thick were prepared and autoradiographs were made io'J by the dipping method using Ilford emulsion. After 3 weeks of exposure at 4°Cin a wooden light-tight box, the slides were developed in Kodak D19 B and fixed. The developed slides were stained with phloxine-hemalum stains. The number of labeled mitoses per 100 total mitoses was plotted against time after the /-> pulse label and the form of the curve was obtained. By this means, E it was possible to determine the duration of the cell cycle which was measured at 60% values on the ascending curves. The mini mum duration of GÃŒisdetermined by the time between the injec 10'. tion and the appearance of the first labeled mitoses. The average duration of G2 is the time between administration of the thymi- dine-3H and when 50% of the mitoses were labeled. The duration of mitoses was determined from the mitotic index multiplied by »I the generation time which was in turn derived from the period between the midpoints of 2 successive waves of labeled mitoses. o The duration of the synthetic period for the in vivo experiment was determined as the time between the 50% labeled mitoses on m o the ascending and descending slopes of the curve. Gìwascalcu 10 _ lated as the difference between the total generation time and the sum of the other phases of the cell cycle. The labeling index (L.I.) was measured in mice killed 1 hour and 5 days after pulse labeling. Furthermore, 6 mice were in ] 1 jected with 50 microcuries thymidine-3H every 5 hours for 30 hours, and the percentage of labeled cells was determined on the tumors of mice sacrificed one hour after the 7th injection. In all experiments, the background in the autoradiographs was ex tremely low and cells containing 2 grains and more were con O 5 10 15 sidered as positive cells. The growth fraction (11) was determined by 2 independent DAYS methods: (a) Five days after a pulse label of thymidine-3H the CHART 1. Growth curve of the NCTC clone 2472 in vivo (C3H ratio between the percent of labeled cells and the percent of mouse). labeled mitoses gives an estimate of the proportion of dividing cells in a total tumor population if certain theoretic conditions set forth by Mendelsohn (11) are satisfied, (fo) Knowing the cell surface area is more than 350 sq ß(circleof diameter, 21 n). duration of the S phase (T¡)and the duration of the cycle (Tc) When the surface area is less than 350 sq /u,growth slows and the we can calculate a theoretic labeling index: plateau is reached when the mean cell surface area is 140 sq fj. (circle of diameter, 13 p). The cell in sus]>ension is a sphere of 14 L.I. = T./Tc /z diameter. In order to have cells which are growing in the exponential If the L.I. observed corresponds to the L.I. calculated, then phase, in the slowing phase and in the plateau at the same time, one can consider that the whole ¡wpulationis proliferating. The it was necessary to stagger the time at which the cultures were ratio L.I. observed/L.I. calculated is equal to the growth frac started. For the exponential phase cells, cultures were started 48 tion. hours before the time of the experiment while for the slowing cells and plateau cells, the cultures were started at 72 hours and In Vitro 96 hours, respectively, before the time of experiment. The num Stock cultures of NCTC clone 2472 were plated into 6-cm ber of cells per culture at the start and in the course of the study Petri dishes with an initial number of 2 X IO5cells per plate.