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 fibrosarcomaâ€”i.e., from the 15th to the 32d day after transplantationâ€”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)Â¿iÂ¿22;d\and Â¿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 mÃ©tastases, 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

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.â€”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 dayâ€”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 dayâ€” 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.â€”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 1F16Fâ€”54FM tumor the 9th day, most of the tumor consisted of necrot (20)â€”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. BERTALANFFYANDLAUâ€”CellDivision in Rat Tumors cause enough well preserved tumor specimens could group for each 6-hour period. The variation in mi not be obtained. totic rate of the animals in each group was ex With fibrosarcoma, sufficient tumor tissue be pressed as the standard deviation. Analysis of vari came available only 15 days after transplantation ance revealed no statistically significant difference of the tumor samples. From this time the tumors among the four means of either tumor (variance grew steadily and rather rapidly until onset of ne between means < variance within groups) and crosis occurring during the 4th or 5th week after thus no diurnal variations. The average mitotic transplantation. Subsequently, necrosis progressed rate of the Walker tumor was estimated to be 15.8 rapidly, often resulting in extensive ulcÃ©ration.It per cent for any 6-hour period of the day, the total was not possible to obtain suitable specimens later standard deviation being 2.37 per cent (Table 1). than 5 weeks after transplantation. Accordingly, The average mitotic rate of fibrosarcoma for any the mitotic rates were estimated within the period 6-hour period of the day was estimated to be 10.7 from 15 days until 32 days after transplantation. per cent (Table 1). The daily mitotic rate (the per To ascertain whether the mitotic rates of these centage of cells formed during one 24-hour period), tumors underwent variations throughout the day, obtained by summation of the four 6-hour per the rates were determined during one complete 24- centages of each tumor, was 63 per cent for Walker hour period; for each tumor, four groups of four tumor, and 42.8 per cent for fibrosarcoma (Ta animals each were given injections of colchicine ble 1). and sacrificed at four different time intervals of the Table 2 shows the results of the mitotic counts day. The results of the mitotic counts are tabu of each tumor on different days after transplanta lated in Table 1. Each one of the four percentages tion. The percentage of the 6-hour period of each shown for each tumor represents the average of the day represents the average of the individual per individual percentages of the four animals in each centages of the four animals composing each group; TABLE 1 MITOTICRATESOFWALKERCARCIXOSARCOMAANDRATFIBROSARCOMADURINGA24-HouR PERIOD Each 6-hour group consisted of four animals

NUCLEIWalker METAPHASESWalker METAPHASESWalkerOF

TIIIE orDAT10 256 Â«56977296979903102841F16F9066917589819112No.25614121536150218221F16F10121030861988Daily MeanÂ± at.dev.14.4Â±2.5S15.8 MeanÂ±jt.dev.11.2Â±0.2011.2+0.289.6

P.M.4A.M.- 4 P.M.10P.M.-10 +2.3215.2 PM- 4AJl.4 +1.6917.7Â±2.4263.115.8Â±2.371F16F+1.8710.8+2.7342.8 A.M.-10 A.M.No.

mitotic rate Over -all average of 6-hour periodsPERCENTAGE 10.7Â±1.70

TABLE 2 MITOTICRATESOFWALKERCARCINOSARCOMAFROMTHESTHTOIOTHDAYANDOFRAT FIBROSARCOMAFROMTHEISTHTOTHE320 DAYAFTERTRANSPLANTATION Each 6-hour group consisted of four animals

AFTERTRANSPLANTATIONWalkerDATS NUCLEIWalker UETAPHASESWalker UETAPBASESWalkerPERCENTAGE OF

256 25656789101F16F151822262882NO.25610082929297729188898988661FI6F948390728932914466959066No.256158614461412118913391289IF16F9599778779476371012Av.:6-HOCR MeanÂ± St.dev.15.7Â±2.2215.6Â±4.3614.MeanÂ±Â»t.dev.10.1Â±0.0710.8Â±0.069.8

4 Â±2.5512.9Â±1.7014.9 +0.1410.4 +0.129.5 +2.3914.5Â±2.5014.7Â±2.861F16F+1.8411.2 +0.2010.3

+ 0.11

the variation in mitotic rate of each group is indi period; tumor growth apparently continued at a cated as the standard deviation. With neither tu steady rate, unaffected by day and night, or by mor did analysis of variance detect any significant sleeping and waking of the animals. A major ob differences among the means of the six days (vari servation was that there was no significant differ ance between means < variance within groups). ence apparent between the mitotic rates of any The average mitotic rate of Walker carcinosar- day during the growth period of either tumor. This coma over any 6-hour period of any day after indicated that both tumors grew at a steady rate transplantation was 14.7 per cent, the total stand from the time the tumor had adapted itself to the ard deviation being 2.36 per cent (Table 2) ; this is host until the mitotic rate fell off with advanced not significantly different from the over-all average necrosis. During active growth, the daily increase of 15.8 per cent obtained in the 24-hour experiment in the number of cells in the tumors remained con (Table 1). The average mitotic rate of fibrosar stant and did not fluctuate appreciably. This ob coma over any 6-hour period of any day was 10.3 servation of constant tumor growth is in agreement per cent, the standard deviation being 0.11 per with a similar finding previously reported by cent (Table 2) ; this is nearly similar to the over-all Schrek (18) on the growth of Walker carcinosar average of a 24-hour period of 10.7 per cent (Ta coma and Flexner-Jobling rat carcinoma. ble 1). The average mitotic rate of Walker carcinosar With the fibrosarcoma, female rats in all stages coma for any 6-hour period during the 5th-10th of the estrous cycle were used. The minimal differ day after transplantation was 14.7 per cent (Table ences of mitotic rates of the animals would imply 2). This indicated a daily cell addition to the tumor that the rate of mitosis of this tumor remained un of close to 60 per cent, or a 100 per cent cell in changed during the estrous cycle. crease every 1.7 days. In fibrosarcoma, the average mitotic rate of any 6-hour period during the 15th- DISCUSSION 32d day after transplantation was 10.3 per cent Growth of a transplanted tumor can be divided (Table 2), indicating a 40 per cent addition of new into three periods: (a) a latent period when the tu ly formed cells daily, or a cell increase of 100 per mor adapts itself to the new host; (b) a period of cent every 2.5 days. These calculations are based growth when the constituent cells actively pro on the assumptions that all cells in the growth liferate and the newly formed cells outnumber by areas counted were capable of cell division and far those lost by degeneration ; and (c) a period of that their interphase was of similar duration. If degeneration when the tumor undergoes extensive this is the case, the figures of 1.7 and 2.5 days, re necrosis and liquefaction. Death of the animal may spectively, also denote the typical duration be shortly follow this latter period. With the Walker tween two divisions. Even though about 15 and 10 carcinosarcoma, the latent period was 4 days after per cent of cells are added to Walker tumor and transplantation, with fibrosarcoma about 2 weeks. fibrosarcoma, respectively, during each 6-hour pe Active growth of the Walker tumor proceeded be riod, it is assumed that most of the newly formed tween the 5th and 10th day; it continued up to 4 cells would not divide during the next 24 hours weeks after transplantation with the fibrosarcoma. (i.e., the duration of a complete colchicine experi Following the growth period was onset of necrosis; mentâ€”Table 1). most animals in the group with Walker tumor had Comparison of the daily mitotic rates of Walker died 2 weeks after transplantation. Rats with fibro carcinosarcoma and fibrosarcoma (60 and 40 per sarcoma rarely survived 7-8 weeks. cent, respectively) with those of normal rat tissues The mitotic rates of Walker carcinosarcoma 256 reveals that their rates of cell formation exceed and of fibrosarcoma 1F16F were determined on those of most organs. The daily mitotic rates of the various days after transplantation during the ac tumors are considerably higher than those of the tive growth period. In neither tumor were fluctua urinary bladderâ€”1.6 per cent (17) and ureterâ€”3 tions in mitotic rate apparent during a 24-hour per cent (4), bronchus and tracheaâ€”2-4 per cent

FIG. 1.â€”Portion of Walker carcinosarcoma 256 from a rat given injection of colchicine. The cells varied greatly in shape and size, and so did their nuclei. Some nuclei contained coarse chromatin particles and large distinct nucleoli. Several "colchi cine metaphases" are visible (e.g., in right lower corner), with clumped, irregularly shaped black chromosomal material. H. & E., X480. FIG.2.â€”Portion of fibrosarcoma 1F16F from a rat given in jection of colchicine. The cells were large, and the nuclei dis played marked anisokaryosis. Scattered among the resting cells were several "colchicine metaphases" with darkly staining clumped chromatin material. H. & E., X420.

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(3), epidermisâ€”3-5 per cent (1, 19), colonâ€”10per results. Technical assistance by Miss C. McAskill and Miss cent (2), esophagusâ€”11 per cent (2), sebaceous R. Mueller is acknowledged. glandsâ€”13 per cent (1), pulmonary alveolar cells â€”15per cent (3, 5, 6), lymphocytes in bronchial REFERENCES 1. BERTALANFFY,F.D. Mitotic Activity and Renewal Rate lymph nodesâ€”15per cent (5), and rectumâ€”16 per of Sebaceous Gland Cells in the Rat. Anat. Ree., 129:231- cent (2). The daily mito tic rate of Walker tumor 41, 1957. is more than twice as high as those of buccal mu 2. . Mitotic Rates and Renewal Times of the Digestive cosaâ€”24 per cent (2), and of anal epidermisâ€”23 Tract Epithelia in the Rat. Acta anat, 40:180-48, 1960. 8. . The Modern Concept of Respiratory Tissue per cent (2). The daily rate of new formation of Structure. Acta Cytol., 6:885-89, 1961. cells in fibrosarcoma is exceeded by that in the 4. BEIÃŽTALANFFY,F.D., and LAU,C. Cell Renewal. Int. Rev. pyloric mucosaâ€”54 per cent (16), and the daily Cytol., 13:357-66, 1962. mitotic rates of both tumors are surpassed only by 5. BERTALANFFY,F.D., and LEBLOND,C.P. The Continuous Renewal of the Two Types of Alveolar Cells in the Lung those in the epithelium (crypts) of the small intes of the Rat. Anat. Ree., 115:515-42, 1953. tineâ€”64-79 per cent, both of the rat (2, 15) and 6. . Structure of Respiratory Tissue. Lancet, II: 1365- man (7). 68, 1955. It may be of interest to compare and contrast 7. BERTALANFFY,F.D., and NAGY,K. P. Mitotic Activity and Renewal Rate of the Epithelial Cells of Human the significance of mitotic activity in normal and Duodenum. Acta anat., 45:362-70, 1961. malignant tissues. The mitotic rates of the malig 8. BLUMENFELD,C.M. Studies of Normal and Abnormal nant tumors exceed those of most normal tissues; Mitotic Activity; Rate and Periodicity of Mitotic Activity however, they are not higher than some mitotic of Experimental Epidermoid Carcinoma in Mice. Arch. rates that may normally occur. During the period Pathol., 35:667-73, 1948. 9. CHAMBERS,H.,and SCOTT,G.M. Variations in Growth of of growth apparently most malignant cells retain Jensen Rat Sarcoma and Influence of Technique. J. Path. the ability to divide. In normal epithelia, chiefly Bact., 33:553-61, 1930. less differentiated cells (such as basal cells) undergo 10. DUBLIN,W. B.; GREGG,R. O.; and BRODERS,A.C. Mito mitosis, and the majority of newly formed cells dif sis in Specimens Removed during Day and Night from ferentiate, for example, into superficial squamous Carcinoma of Large Intestine. Arch. Pathol., 30:893-95, 1940. cells, columnar secretory and absorptive cells, etc., 11. EDWARDS,J.L.; KOCH,A. L.; Youcis, P.; FREESE,H. L.; which, after having reached maturity, divide rare LAITE,M. B.; and DONALSON,J.T. Some Characteristics ly or not at all. In both malignant tumors a con of DNA Synthesis and the Mitotic Cycle in Ehrlich As- stant number of cells divide daily. Similarly, in cites Tumor Cells. J. Biophys. Biochem. Cytol., 7:273-82, 1960. many adult normal tissues the numbers of cells 12. EIGSTI, O. J., and DUSTIN,P. Colchicine in Agriculture, that each day undergo division are constant. How Medicine, Biology and Chemistry. Ames, Iowa: Iowa ever, in the latter mitosis serves cell renewal, State College Press, 1955. and for each newly formed cell another, usu 13. GAYLORD,H.R., and CLOWES,G. H. A. On Spontaneous ally more differentiated cell, becomes extruded; Cure of Cancer. Surg. Gynec. Obst., 2:638-58, 1906. the numbers of cells thus remain constantâ€”or in a 14. KILJUNEN,A. Mitotic Activity in Normal and Malignant Epidermal Tissue of the Rat. Acta pathol. et microbio!. steady stateâ€”in spite of an often considerable mi Scand. (Suppl.), 112:1-101, 1956. totic activity. In contrast, in malignant tumors 15. LEBLOND,C. P., and STEVENS,C. E. The Constant Re mitosis results in cell addition (or accumulation)â€” newal of the Intestinal Epithelium in the Albino Rat. Anat. Ree., 100:357-78, 1948. i.e., in growth of the tumor; thus, about 60 per 16. LEBLOND,C.P., and WALKER,B. E. Renewal of Cell Pop cent of new cells are added each day to Walker ulations. Physiol. Rev., 36:255-76, 1956. carcinosarcoma, about 40 per cent to fibrosarcoma. 17. LEBLOND,C. P.; VULPE,M.; and BERTALANFFY,F.D. Cell loss may occur also from malignant tumors, as Mitotic Activity of Epithelium of Urinary Bladder in Al by necrosis or cell desquamation. Cells are shed bino Rat. J. Urol., 73:311-13, 1955. 18. SCHREK,R. A Quantitative Study of the Growth of the particularly from epithelial tumors, and this is also Walker Rat Tumor and the Flexner-.Iobling Rat Carcino the basis of exfoliative cytology. However, unlike ma. Am. J. Cancer, 24:807-22, 1985. normal tissues, cell formation and cell loss are not 19. STOREY,W. F., and LEBLOND,C. P. Measurement of the balanced in an orderly fashion in malignant tu Rate of Proliferation of Epidermis and Associated Struc tures. Ann. N.Y. Acad. Sci., 63:537-45, 1951. mors, and during the period of growth cell loss is 20. WALLACE,A.C. Metastasis as an Aspect of Cell Behavior. usually exceeded by cell addition. Proc. 4th Can. Cancer Research Conf., 4:189-65. New York: Academic Press, 1961. ACKNOWLEDGMENTS 21. WOGLOM,W. H. Regression of Jensen Rat Sarcoma. J. The authors are grateful to Dr. A. C. Wallace, Cancer Re Cancer Research, 9:171-89, 1925. search Laboratory, University of Western Ontario, London, 22. WÃœST,G.ÃœberdenNucleinsauregehaIt bÃ¶sartigermensch Ontario, for his kind advice and assistance; Dr. Wallace also licher Tumoren und ihrer Metastasen. Arch. Gesch made available the tumor material for the transplants. We are wulstforsch., 16:324-85, 1960. indebted also to Prof. I. Maclaren Thompson, Chairman of 23. . Ãœberden NucleinsÃ¤uregehalt bÃ¶sartigermensch this department, for his kind assistance with the preparation licher Tumoren und ihrer Metastasen. Ibid., 17:10-15, of the manuscript and with the statistical evaluation of the 1960.

Felix D. Bertalanffy and Chosen Lau

Cancer Res 1962;22:627-631.

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