Kinetics of Cell Proliferation of an Experimental Tumor

(CANCER RESEARCH 27 Part 1, 1122-1131,June 1967]

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'.Â¿.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

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 4Â°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 thymidine-3H and when 50% of the mitoses were labeled. The duration of mitoses was determined from the mitotic index multiplied by Â»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. GÃ¬wascalcu 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 ÃŸ(circleof diameter, 21 n). duration of the S phase (TÂ¡)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 Â¡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. The are shown in Chart 3. usual culture medium was used (Medium 109 with 10% added Thymidine-'H (specific activity, 8.5 c/mmole) was added to horse serum). The Petri dishes were placed in an atmosphere of each of the culture dishes to a final concentration 2 juc per ml of sterile air of saturated humidity with 109Â¿added COi in an air medium. The cells were left in radioactive medium for 15 minutes, tight box. The culture medium was renewed every 24 hours to the medium was removed, the cells washed, and new iionradio- avoid the influence of an impoverished medium on the slowing active medium was added. At each time point at which the label down of the growth rate. It is calculated that under these condi ing index of mitoses was to be measured, 2 Petri dishes from each tions the cellular growth rate remains exponential until the mean set, i.e., exixmential, slowing, and plateau cells, were trypsinized,

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1967 American Association for Cancer Research. E. Frindel, E. P. Malaise, E. Alpen, and M. Tubiana the colls were collected, horse serum was added to inactivate the TABLE 1 trypsin, and the cells were centrifuged. The bulk of supernatant Percent Labeled Mitoses at Various Times after the Injection of Thymidine-'H medium was removed and smears were made of cells suspended in a few drops of remaining medium. The slides were fixed in For the 3-day and 7-day tumors, 4 tumors were used for each point and a total number of 50-100 labeled mitoses were counted absolute methanol and dried. Autoradiographs were stained in randomly in these tumors. For the 20-day tumor, a total number 10% Giemsa stain. of 100mitoses was counted in 2 to 3 individual tumors. Asterisk (*) The percentage of labeled mitoses at the various times was indicates labeled mitosis. determined by examination of 50 random mitoses. However, for the plateau cells, mitoses are so infrequent that only 25 mitoses tumorMÂ«01350505050505050505050505050Total-daytumorM'050100100100102102100100100100100100100100TotaltumorM*35232074719693979810085886255584147415857678667808079727874676488878384885384Total Timeafter were examined. Since the background grain count was less than injection (te)15 0.1 grain j>ercell, a mitosis was judged |x>sitive if it had 3 or more M100100109716454691281218284119795773%MÂ«013407178927230416160426388697M100365169121116111135233244178116194150145161Â»0145983869276434156865267696220-dayM100100100100100100100100100100100100100115100100100100100100100100100100100100100100100107115111100100100100100100100%M-05232074719693979810085885455584147415857678667808079727869585888878384885384 grains above it. The labeling index was determined by count ing the number of labeled nuclei in 200-400 cells. The same grain min123571013162024283034405060723-day count criterion was used, i.e., 3 grains per cell and more as ]>ositive. The mitotic index was determined by counting the number of mitoses seen in 2000 cells. These estimates were made on auto- radiograph |Â¡rÃ©parations.

RESULTS

In Vivo The Growth Curve (Chart 1). This is measured from the 6th day. It is possible to calculate the volume of cells injected since the number of cells (750,000) and mean cell volume (1,400 n) are known. The growth curve between the injection and the 6th day is calculated by interpolation. The tumor-doubling time, which can be calculated for each ]K)int by taking the e.\]>onential tangent to the growth curve, increases exponentially with the age of the tumor. The results are compatible with a growth pattern following a Gompertz function (5, 6, 10). The Cell Cycle. The detailed results of the labeled mitosis count are given in Table 1. Two waves of labeled mitoses were obtained in all tumors. The peak was reached by 7 hours and was equal to about 90 to 98%. Chart 2 shows that the cell cycle varies slightly, if at all, with the age of the tumor. The cell cycle of the 3-day tumor is about 16.5 hours (Table 2), and about 17.5 hours for the 7-day tumor and the 20-day tumor. The rt |>eriod is the same for the 3- and 7-day tumor and is equal to about 10 hours. For the 20-day tumor, the S period is about 12.5 hours, a value not significantly different from the others. The labeling index varies only slightly, from 26% for the 3-day tumor to 24% for the 7-day tumor and 20% for the 20-day tumor when the cells are examined one hour after pulse labeling. Five clays after pulse labeling, the labeling index is 22%, 12%, and 10% for the 3-, 7-, and 20-day tumors, respectively. The median number of grains per cell was 27-28 for the 3-day and 33-34 for the 7-day tumors. For the 20-day tumors, the median grain count per cell varied from 10 in fields of low labeling index to 65 in fields where the labeling index was high. The random median grain count )x?rcell was 36 and equal to the rnidrange (Charts 5, 6). The mitotic indices in the 20-day tumors paralleled the labeling index. Table 3 shows the relationship after multiple in jections between the mitotic index and the labeling index in 35 fields. The Mitotir Index (Table 3). The mitotic index of the 3-day tumor is 0.84%; at that time, all the tumors are not yet very well organized as solid, palpable tumors. Four days after injection of the cells, the mitotic index is 3%. It then falls to 1.7% at 7

1124 CANCER RESEARCH VOL. 27

3 DAY TUMOR 100- â€”20 7 DAY TUMOR

O 10 20 30 40 50 60 70 HOURS CHART 2. Cell cycle in vivo of NCTC tumors at various times after implantation. days and to 1.4% at 20 days. The mitotic index at 21.5 days is TABLE 2 0.8%. Growth Parameters in Various Growth Phases of Tumors in Vivo The Growth Fraction. Table 4 gives the results of the growth fraction of the tumors by 2 different methods. There is little (%)1 diffÃ©rencebetween the 3-day and 7-day tumors. By Method 1, time the growth fraction was found to be 40% and 35% for 3- and 7- tumor(days)3472021.5Doubling(hours)24 (%)0.8431.71.40.8Cycle(hours)16.517.517.5Labelingindex hour 7lions848336 day tumors, respectively, and 24% for the 20-day tumors. By afterinjec -Method 2, the growth fraction is 44% for the 3-day tumors, 40% tion202420After for the 7-day tumors, and 29% for the 20-day tumors. With both methods, the growth fraction of the 20-day tumors is lower than (calculated)38 in the younger tumors. This difference is especially emphasized (measured)110 by the multiple injections method. By this method, the labeling (measured)index index is 84% for the 3-day tumor, 83% for the 7-day tumors, and 36% for the 20-day tumors (Table 2). The photomicrographs of multiple injected tumors demon strate the extreme heterogeneity of the late tumors in respect to DISCUSSION the labeling and mitotic indices. It can be seen that some fields Cell Cycle have a high labeling index (up to 82% cells labeled) and high The comparison of curves of labeled mitoses suggests that the grain counts ]>ercell, whereas other fields are very slightly labeled cycle has essentially identical characteristics, whether the cells (only 3% of the cells showing grains) (Figs. 1,2). are growing in an animal host or in the glass system. It also apireara that the cellular cycle remains unchanged as cellular In Vitro proliferation slows either for the in vitro or in vivo growth of the The data on the in vitro growth curves including exixmential tumor. The duration of the phases of the cellular cycle seems also to remain constant for the NCTC 2472 cell line in spite of phase, slowing down phase, plateau, and doubling times are variations in conditions of growth which are important. However, given in Chart 3. The overall results are comparable to those even though the curves apirear to be essentially the same, one obtained for the in vivo growth curves. The labeled mitosis cannot completely exclude a certain small variation in the curves are given in Chart 4, for cells growing exponentially on the duration of some phases. The duration of G2, determined from the slowing phase and at the plateau of growth. The generation time increase in the curve of labeled mitoses, appears to have a parameters derived from these curves (Table 5) are the same maximum value not exceeding 3 hours since the maximum and are comparable to those found in vivo. plateau approaches nearly 100% at this time. Evaluation of the

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duration of S from the length of the plateau of the mitotic curve leads one to conclude that there may be an appreciable variation among individual cells for this phase of the cycle since the slope of the descending portion of the curve is not steep and the curve TIME - 2< HOURS does not fall to low values. However, the variability of S as well as the mean value of S appears to lie analogous for the different curves. It must be reiterated, however, that with the labeled mitoses technic used in these experiments, it is difficult to recog nize the existence of a small proportion of cells having either long or short S phase. The Gj phase seems to have a variable duration, since the return of the curve to a second plateau is not sharp and definitive and the second peak fails to reach a value approaching 100%. Further, the shape of the second wave of labeled mitosis is not the same, the slope being slower for the 20-day tumor curve. This may indicate, in these tumors, a larger 10 fluctuation in the duration of the GÃ¬phase, the mean deviation of which may be slightly prolonged. To rule out the possibility TIME IN DAYS that a small proportion of cells has either a very long or a very CHART 3. Growth curve of the XCTC clone 2472 in vitro. short cell cycle, it would be necessary to undertake more com-

100-

80-

(O otâ€” i o

EXPONENTIAL GROWTH 20 SLOWING GROWTH PLATEAU

O 2 5 7 10 12 16 20 24 HOURS

CHART 4. Cell cycle in vitro of NCTC cells during the exponential growth, the slowing growth, and the plateau.

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7-DAY TUMOR: MEDIAN GRAIN COUNT i 20 GRAINS PER CELL

bJ U

O 30

OC Ul IO z ID z 20

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25 50 75 GRAINS PER CELL CHART5. Histogram of grain counts in 7-day tumors. plicated experimental designs, for example the application of the examine the proi>ortion of the cells engaged in reduplication and technics of double labeling, but in any case, whether this phe division. The growth fraction may be examined by several inde nomenon exists or not, it does not apirear to have sufficient pendent technics for the cells grown in vitro. The first of these importance to explain the slowing of tumoral growth. Men assumes that the time for mitosis remains the same in spite of delsohn (12) found cell cycles of the same duration in 143 variation of the fraction of cells in the growth phase. If this tumors of different ages. Baserga and Gold (2) found in an Ehrlich assumption is valid, the ratio of mitotic indices in various growth ascites that the S and GÃŒphaseswere practically the same in the phases is also the ratio of their growth fractions. The calculations various stages of the ascites growth. Our results corroborate lead to the results shown in Table 6. It is also [x>ssibleby the same those findings. It seems likely that the duration of the cell cycle reasoning to estimate the growth fraction by the ratio of the is a characteristic of the type of cell and does not change notice labeling index to the measured fraction of the generation time ably during the evolution of a tumor. This is confirmed by the occupied by synthesis (T,/TC). These calculations are also shown in vitro studies. in Table 6. It is apparent from the data in Table 6 that, except for the In Vitro exponential phase of growth, an increasingly smaller fraction of As in the growth of this tumor cell line in vivo, one cannot the cells are engaged in synthesis or division. However, even account for the rapidly altering growth rate on the basis of al more important, a large discrepancy exists between the relative tered chronology of events in those cells which are, in fact, birth rates of cells as estimated from the kinetic parameters and in the process of reduplication and division. The time between the relative birth rates as measured by increase in cell numbers divisions is 17 hours, almost exactly the same as the time found as in Chart 3. Even in the exponentially growing system, there is for the same generative cycle in tumor growth in vivo. This a deficit between the number of cells actually produced and the generation time is also slightly shorter than the doubling time number estimated. In the more slowly growing cultures, a great for the number of cells during e\i>onential growth phase in vitro percentage of the calculated production does not contribute to (18 hours). the increase in cell numbers and are lost. Since all the kinetic Since the cycle is not significantly altered, it is necessary to parameters are internally consistent and the ratio of labeling

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50- 20 DAY TUMOR : FIELD OF LOW LABELING INDEX MEDIAN GRAIN COUNT 10 GRAINS PER CELL

20 DAY TUMOR FIELD OF HIGH LABELING INDEX MEDIAN GRAIN COUNT: 65 GRAINS PER CELL

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25 50 75 100 GRAINS PER CELL CHART6. Histogram of grain counts in 20-day tumors. index to mitotic index remains essentially constant, the results of TABLE 3 this calculation seem to be valid. For the plateau culture, the Relationship after Multiple Injections between Mitotic Index and calculation indicates that roughly 1.6% of the cells are disappear Labeling Index in W-day-old Tumors ing per hour from the culture. It is interesting to note that if the medium is renewed fre No. offields236321No. ofmitoses01234% Labeledcells2039536364 quently, the plateau is reached when each cell fixed to the glass takes up a surface area corresponding to a circle of 12 judiameter. This is almost the same as the diameter of the cell in sus]>ension (14 fj.).One can therefore understand how under these conditions the number of cells attached to the wall cannot increase, espe cially as this cell type only grows as a monolayer in vitro.

In Vivo knowing the doubling time (taken as the exponential tangent of We have also found in vivo that in the course of tumor growth, the growth curve) and hence the observed increment of the num there is a decrease in growth fraction. The decrease in the number ber of cells it is easy to calculate the proportion of cells which of labeled cells after multiple injections is especially noticeable. must be engaged in the cell cycle to give rise to the requisite num However, this decrease does not seem to be quite sufficient to ber of cells. The ratio of the number of the cells in these 2 com account for the slowing of tumoral growth, with any of the partments is equal to a theoretic growth fraction. In order to models of growth pattern (exponential or Gom]>ertzian) used for account for the change in doubling time from 27 to 110 hours in the calculations. We have indicated in Table 3 the theoretic the course of tumoral growth, one must accept a variation in this values of the growth fraction which are calculated assuming a theoretic growth fraction which is as much as a factor of 6. On the simple 2-compartment model in which the cell population is com basis of the simple model put forward above, the growth fraction posed of cells at rest or engaged in a 17-hour cycle. It is further must, for instance, vary during the interval from the 7th to the assumed that all mitoses give birth to two viable cells. Thus 2()th day between 35% and 10%. The variations in growth frac-

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TABLE 4 TABLE 6 Growth Fraction and Kinetic Parameters for KCTC Tumor in Vivo Growth Fraction Calculations for NCTC Cells Grown in Vitro

tumor4044002.102.557-daytumor3540351.801.8020-daytumor242910l.GOO.GOcalculatedGrowthParameter fractionMethodGrowth fractionL.I.T./Te(Irowth 1:L. I.(M*/M) 100Method X fractionM.I./M.I. exp.Birth 2:L.I.Tt/TcTheoretic rateL.I./F,Birth

rateM.I./TMRatio 2%/hour220.3%/hoiirPlateau27%26%1.6%/hour1.6%/hour230 (2compartments:growth fraction L.I./M.I.Increment 17hours)Birth cell cycle, ofthecell number(measured)Exponential86%5%/hour6.4%/hour194%/hourSlowing31%34%2%/hour2. rate(L.I./21,)Increment " T,, duration of phase S; Te, duration of cycle; M.I., mitotic number(measured)3-dayof the cell index; M.I. exp., mitotic index during exponential phase; TÃŒI, duration of mitosis; L.I., labeling index.

(M*/M) X 100, percent of labeled mitoses; T., duration of small increase to only 30% after 7 injections every 5 hours in 20- phase S; Te, duration of cycle; L.I., labeling index; asterisk (*) day tumors. Even if one accepts that those cells which are not in indicates labeling. the S phase at the time of the first injection never divide, the increase in the labeling index should be clearly larger than that TABLE 5 observed. Another explanation could be a synchrony of cells in Generation Time Parameters, Mitotic Index, and Labeling Index old tumors, but this explanation seems improbable. This con for NCTC Cells in Various Growth Phases in Vitro flict in our results suggests as in the case of the culture in vitro (hours)"To.2.7523T.121312TGI+ÃŒI2.2522Te171717Mitoticparameters that a non-negligible pru)H>rtion of cells disap|x>ar immediately time after mitosisâ€”either dying or migrating from the tumor. Fur phaseExponentialSlowingPlateauCycleGrowth index(%)612419Doublingof cell index(%)3.21.10.83Label-.'"/number (hours)1824OCthermore, comparison between calculated relative birth rate and relative increase in cell number observed on the growth curve shown in Table 4 ap|x>ars to confirm this latter hypothesis. The mitotic index for 20-day tumors is 1.4%. While this is certainly inferior to the maximum value of 3'/Â¿seen for the young tumors " T,, duration of phase S; Tc, duration of cycle; ToÂ»duration and for tumor cells in vitro, it does suggest again that a signifi of phase pulation of cells which are at rest, and a of cells labeled varies considerably from one region to the other third population of cells which may have prolonged cell cycles of in the tumor. The slowing of growth is thus not a universal greater than 17 hours, one can adequately account for the changes process throughout the whole tissue mass but is very hetero in tumor growth and labeling indices. Hut these calculations have geneous. It seems that certain regions of the tumor continue to not been extended since by their characteristics they are arbi proliferate rapidly, while in others, nearly all the cells are found trary. We are now in the process of studying cell cycles by the at rest. One may, of course, ask if such a result is not due to an double isotoix; technic in order to substantiate this model. In artifact, the labeled precursor not reaching certain regions of the summary, there is certainly a diminution of the growth fraction tumor. This is an unlikely explanation since the mitotic index but we cannot exclude the )Â»ssibility that certain phases of the and the labeling index vary in a parallel fashion throughout the cell cycle as GÃ¬and S may be prolonged somewhat. Another phenomenon may provide an explanation of the ob tumor. Analogous observations have been made by certain other served facts and allows at the same time an interpretation of the authors on mice (4) or human tumors (3), in which it has been other observations which cannot be explained by a prolongation observed that there exist regions in which there are practically of the cell cycle. One must find an explanation for the contract no cells labeled. These variations have in general been attributed between the labeling index of 20% after a single injection and the to variability in conditions of vascularization; however, we have

JUNE 1967 1129

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1967 American Association for Cancer Research. E. Frindel, E. P. Malaise, E. Alpen, and M. Tubiana not observed that the regions which are heavily labeled in the 2. Baserga, R., and Gold, R. The Uptake of Tritiated Thymidine mouse tumor have a particularly marked increase in blood sup by Newly Transplanted Ehrlich Ascites Tumor Cells. Exptl. Cell Res., 3/.- 576-585, 1903. ply over those regions which are less well labeled. Thus, we can 3. Kissel, P., Duprez, A., Schmitt, J., and Dollander, A. Auto- not confirm a hypothesis of variability in vascularization as a historadiographie des Cancers Digestifs Humains in Vivo. basis for inhomogeneity of labeling, but our results do not on the Compt. Rend. Soc. Biol. 159: 1400-1403, 1905. other hand permit us to exclude it. It must be remarked, how 4. Kligerman, M., Heidenreich, W. F., and Greene, S. Distribu ever, that, as in man, the mean grain count is lower in the region tion of Tritiated Thymidine About a Capillary Sinusoid in a where labeled cells are infrequent than it is for those regions Transplanted Mouse Tumor. Nature, 196: 282-283, 1962. where the labeling index is high. The mean number of grains per 5. Laird, A. K. Dynamics of Tumor Growth. Brit. J. Cancer, 18: cell in a high labeling index region is comparable to that found 490-502, 1964. for cells of tumors which are growing rapidly while the poorly 6. Laird, A. K. Dynamics of Tumor Growth: Comparison of labeled regions have mean grain counts of less than Ã¬ofthe higher Growth Rates and Extrapolation of Growth Curve to One Cell. Brit. J. Cancer, 19: 278-291, 1965. value. One may interpret the variation in grain count per cell as either the result of variability in vascularization or by a varia 7. MacQuilkin, W. T., Evans, V. J., and Earle, W. R. The Adap tation of Additional Lines of NCTC Clone 929 (Strain L) tion of the synthetic rate for DNA. Cells to Chemically Defined Protein Free Medium NCTC 109. The variation in labeling index from one zone to the other in J. Nati. Cancer Inst., 19: 885-908, 1957. the tumor without a corresponding variation in histologie aspect 8. Malaise, E., and Tubiana, M. Croissance des Cellules d'un raises a problem of significance relative to the growth fraction. Fibrosarcome Experimental IrradiÃ© Chez la Souris C3H. The cells which do not divide, are they in G0,if so is this state Compt. Rend. Acad. Sci., 26S: 292-295, 1966. reversible? Also, may the cells be stimulated to re-enter in divi 9. Malaise, E., Tubiana, M., and Barski, G. Nombre de Chromo sion by appropriate external influences? Baserga and Gold's somes et RadiosensibilitÃ© des Tumeurs ExpÃ³rimeiitales. J. data (2) showing that Ehrlich ascites tumor cells at a plateau Radio!. Electrol., 45: 101-105, 1964. stage would promptly re-enter DNA synthesis when transferred 10. McCredie, J. A., Inch, W. R., Kruuv, J., and Watson, T. A. The Rate of Tumor Growth in Animals. Growth, %9: 331-347, to new mice and our own results on tumor growth after irradiation (8) suggest that probably a high proportion of cells are in a 1965. 11. Mendelsohn, M. L. Autoradiographic Analysis of Cell Pro reversible state and can be stimulated to re-enter division. liferation in Spontaneous Breast Cancer of C3H Mouse. III. The Growth Fraction. J. Nati. Cancer Inst., S8: 1015-1029, ACKNOWLEDGMENTS 1962. We are indebted to Mrs. FranÃ§oise Vassort for help and dis 12. Mendelsohn, M. L. The Kinetics of Tumor Cell Proliferation. cussion and to Dr. R. Gerard Marchant and his staff, for their In: M. D. Anderson Hospital and Tumor Institute (eds.), work on the pathology of the tumor. We would also like to thank Cellular Radiation Biology, pp. 498-513. Baltimore: The Miss Ruzica Marianovitch, Mrs. Gilberte Grange, Mrs. Nicole Williams & Wilkins Co., 1965. Chavaudra, and Mrs. Nicole Moreau for their technical assistance. 13. Sanford, K. K., Likely, G. D., and Earle, W. R. The Develop REFERENCES ment of Variations in Transplantability and Morphology 1. Baserga, R. The [Relationship of the Cell Cycle to Tumor Within a Clone of Mouse Fibroblasta Transformed to Sarcoma- (Â¡rowth and Control of Cell Division: a Review. Cancer Res., Producing Cell in Vitro. J. Nati. Cancer Inst., 16: 215-238, 35: 581-595, 19C5. 1954.

FIG. 1. Photomicrograph of a multiple-injected 20-day tumor showing heterogeneity with respect to the labeling index. X 900. FIG. 2. Photomicrograph of a multiple-injected 20-day tumor showing heterogeneity with respect to the labeling index. X 144.

1130 CANCER RESEARCH VOL. 27

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JUNE 1967 1131

Emilia Frindel, Edmond P. Malaise, Edward Alpen, et al.

Cancer Res 1967;27:1122-1131.

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