EFFECT OF THE SIZE OF INOCULUM ON THE GROWTH OF TRANSPLANTABLE RAT TUMORS 1

Transcription

1 EFFECT OF THE SIZE OF INOCULUM ON THE GROWTH OF TRANSPLANTABLE RAT TUMORS 1 ROBERT SCHREK, M.D. (From the Department 01 Pathology, Vanderbilt University School 01 Medicine, Nashville, Tennessee) A knowledge of the effect of technical factors on tumor growth is necessary for the -interpretation of experimental findings on tumor growth. Among factors that should be considered are the age, sex, strain and nutrition of the animal inoculated, the size and character of the inoculum, the site of inoculation, and the normal variability of the measurements of tumor growth under standard conditions. The effect of the size of the inoculum is considered in this paper. A few investigators have studied the effect of the size of the inoculum on tumor growth. Bashford, Murray, Haaland, and Bowen (1) realized the importance of the dose on the subsequent growth of the tumor. Their results varied with different tumors and with the same tumor at different times. In some cases the tumors grew with a rapidity proportional to the initial dose. In most cases, however, small doses grew progressively whereas large doses, even when followed by a more pronounced initial proliferation, gave tumors which remained stationary or disappeared spontaneously. According to these investigators, the variations in the behavior of the same tumor indicated that the tumors are biologically different at different times. Koenigsfeld and Prausnitz (2) found that tumors developed in all mice inoculated with a 1: 5 and a 1: 25 dilution of a tumor suspension, but only one tumor developed in 5 mice inoculated with a 1:3125 dilution of the same suspension. They also found that the smaller the inoculum, the later the tumor started to develop. Frankel (3), working with a small number of chickens, observed 100 per cent takes of the Rous tumor with large inocula, no takes with small inocula, and varying results with those of intermediate size. He also observed that the greater the number of cells injected, the quicker the Rous sarcoma developed. Costa (4) was not able to confirm Koenigsfeld and Prausnitz' observations that the smaller the number of inoculated tumor cells, the longer the period before the tumor develops. MacDowell (5) found that the larger the number of leukemia cells inoculated, the smaller was the percentage of survivors in a pure mouse strain. He also showed that the larger the inoculum, the more rapidly the animals died of leukemia. Shear (6) titrated a number of mouse tumors in pure mouse strains and found that the larger the inoculum, the greater was the percentage of takes and the briefer was the latent period. His results also showed that the suspensions of the various tumors differed markedly in their potency or titer. 1 Aided by a grant from the International Cancer Research Foundation. Received for publication June

2 TRANSPLANTABLE RAT TUMORS 365 In a previous publication (7) it has been shown that the percentage takes of Walker tumor 256 and the Flexner-jobling carcinoma varied directly with the size of the inoculum. Furthermore, the growth of these tumors was analyzed into two growth components: a latent period and a growth constant. The size of the inoculum was found to have a marked effect on the latent period but no perceptible effect on the growth constant. More recent work (8) indicated that a third growth component has to be considered in analyzing the growth of immunizing tumors such as R39 sarcoma. These R39 sarcoma studies have necessitated further observations on the effect of size of inoculum on tumor growth. The tumors used in this study are R39 sarcoma, Walker carcinoma 256A, Flexner-Jobling carcinoma and carcinoma 256B. The tumor 256B was derived from tumor 256A, as will be described in a subsequent publication (9). EFFECT OF SIZE OF INOCULUM ON PERCENTAGE OF TAKES 2 Numerous suspensions of four rat tumors were titrated in rats by the injection of 0.1,0.01 and c.c. amounts. The results of these titrations are summarized in Table 1. As can be seen from the table, the injection of 0.1 c.c, TABLE I: Effect of Size of Inoculum on Percentage of Takes of Four Rat Tumors 0.1 c.c, Inoculum 0.01 c.c. Inoculum c.c, Inoculum Tumor No. of No. of Per- No. of No. of Per- No. of No. of Persus- rats cent- sus- rats cent- sus- rats centpen- inocu- age of pen- inocu- age of pen- inocu- age of sions lated takes sions lated takes sions lated takes B R A Flexner-Jobling amounts of the suspensions produced tumors in 91 to 100 per cent of the rats; 0.01 c.c. produced S6 to 99 per cent takes; and c.c. resulted in 20 to 90 per cent takes. The results with these four tumors show that decreasing the inoculum decreases the percentage of takes. There did not seem to be any marked differences in the susceptibility of the rats to the four tumors. They appeared to be most susceptible to tumor 2S6B (90 per cent takes with c.c. inocula) and least susceptible to tumor 2S6A and Flexner-Jobling carcinoma (34 and 20 per cent takes with c.c.), To obtain any definite conclusions on relative susceptibility to the four tumors, it would be necessary to determine the concentration of the viable tumor cells in each of the cell suspensions used. 2 The percentage of takes was obtained by dividing 100 times the number of rats developing progressive or regressive tumors by the number of inoculated rats that survived at least fourteen days.

3 366 ROBERT SCHREK EFFECT OF SIZE OF INOCULUM ON AVERAGE LATENT PERIOD A comparison of the frequency curves of the latent periods of tumor 256B developing from 0.1, 0.01 and c.c. inocula is given in Fig. 1. The graph was constructed on arithmetic probability paper described in the previous report (8). The graph shows that the three frequency curves are entirely distinct and that the mean latent period of tumors developing from 0.1 c.c. inocula was 2.2 days; from 0.01 c.c., 4.0 days; and from c.c., 8.6 days. The graph indicates further that the frequency distributions of the latent periods for the tumors developing from 0.1 and 0.01 c.c, inocula are normal and can be represented by straight lines, whereas the frequency distribution corresponding to c.c. inocula is abnormal and can be represented only DAYS 16 -r :-""':":"--:-=~:""""'i / 00",",",,"CO" MEAN 10 II 6... Z LoJ... ~ O+-T---.-.Ly--_r r_...i--t~~-_r_..,...-t~ j I 2 S SO eo 90 9S PER CENT CUMULATIVE FREQUENCY - LESS THAN FIG. 1. FREQUENCY CURVES OF THE LATENT PERIODS OF TUKOJIS 256B DEVELOPING FROK OJ, 0.01, AND C.C. INOCULA by a curve. A review of the methods used suggests that this abnormality in frequency distribution may be the result of a slow deleterious action on the tumor cells by the diluent (physiological saline) used for the preparation of the tumor cell suspensions. The statistical constants of the latent periods are compared in Table II. The average latent periods of the tumors developing from 0.1 c.c. inocula were 2.32, 2.42, 1.99 and 3.14 days; from 0.01 c.c. were 4.03, 4.77, 7.06, and 7.53 days, and from c.c. were 9.09, 7.69, and days. The differences in the average latent periods for the three inocula ranged from 1.71 to 7.97 days. These differences were undoubtedly significant, as the probable errors of the differences were 25 per cent or less. It may be concluded that for all four tumor strains, the smaller the inoculum, the greater was the average latent period.

4 TRANSPLANTABLE RAT TUMORS 367 TABLE II: Effect of Size of Inoculum on AfJerage Latent Periods of Four Rat Tumors Tumor Differencest in 0.1 c.c. Inoculum 0.01 c.c, Inoculum c.c, Inoculum average latent periods No. Average No. Average No. Average 0.01 and and of latent of latent of latent 0.1 c.c, 0.01 c.c, tumors period tumors period tumors period inocula inocula days days days days days 256B ± ± ± ±10% 5.06± 6% R ± ± ± ±10% 2.91±12% 25M ± ± ± ±12% 7.97±19% Flexner- Jobling ± ± ± ±18% 7.37±25% Probable error. significant. t Differences with probable errors less than 33 per cent are statistically EFFECT OF SIZE OF INOCULUM ON AVERAGE GROWTH CONSTANT A summary of the effect of the size of the inoculum on the growth constant is given in Table III. Except for the average growth constant of the three TABLE III: Effect of Sile of Inoculum on the AfJerage Growth Constant of Four Rat Tumors Tumor Dllferencest In O.I c.e. Inoculum 0.0 I c.c, Inoculum 0.00 I c.c, Inoculum average growth constants No. Average No. Average No. Averate 0.1 and 0.01 and of arowth of growth of arowt 0.01 c.c, c.c. rats constant rats constant rats constant Inocula Inocula...lda'Y... lda'y.../d<j'y 256B :1: :1: :1: :1:20% 0.046±72% R :1: :1: :1: :1:29% 0.039:1:146% 256A :1: :1: :1: :1:35% 0.067:1: 87% Flexner Jobling :1: :1: :1: :1:52% :1: 56% Probable error. t Differences with probable errors less than 33% are statistically significant. Flexner-Jobling tumors arising from the c.c. inoculum, there is a slight but consistent decrease in the average growth constant with decreasing inocula. The differences between the average growth constants of the tumors developing from 0.1 and 0.01 c.c. inocula are for carcinoma 256B; for R39 sarcoma; for tumor 256A; and for the Flexner-jobling tumor. These differences are slight, representing a decrease of 7 to 12 per cent in the growth constants. The determination of such slight differences and the demonstration of their statistical significance require large numbers of tumors. In a previous study (7), the number of animals used was not sufficient to demonstrate these slight differences. In the present work, the

5 368 ROBERT SCHREK numbers of rats (122 and 180) used for two of the tumors (256B and R39) were adequate to establish slight differences ( per cent probable error and per cent). The numbers of rats (53 and 36) inoculated with tumor 256A and Flexner-Jobling carcinoma were insufficient to demonstrate significant differences ( per cent probable error and per cent). It is probable that if larger numbers of animals had been used for these two tumors, the differences in the average growth constants would also be found significant. The differences in the growth constants of the tumors developing from 0.01 and c.c. inocula are very slight (0.039 to 0.067), representing a decrease of only 2 to 6 per cent in the growth constants. These differences are not statistically significant. However, the fact that these differences have been observed for three of the four tumors suggests that decreasing the inoculum from 0.01 to c.c. does decrease very slightly the average growth constant. It may be concluded that the size of the inoculum has a slight effect on the average growth constants of these four tumors. EFFECT OF SIZE OF INOCULUM ON PERCENTAGE OF REGRESSIONS 3 AND INHIBITION CONSTANT The effect of the size of the inoculum on the percentage of regressions of the four tumors is shown in Table IV. The percentages of regressions of the TABLE IV: Effect of Sise of Inoculum on Percentage Regressions of Four Rat Tumors 0.1 c,c, 0.01 c.c, c.c, Xl test for Total Inoculum Inoculum Inoculum association between size of Tumor Per- Per- Per- Per- inoculum No. cent- No. cent- No. cent- No. cent- and perof age of of age of of age of of age of centage tumors regres- tumors regres- tumors regres- tumors regres- regressions sions sions sions sions P*= R Flexner- Jobling A * High P values (over 0.05) indicate no appreciable association between size of inoculum and percentage regressions. tumors developing from 0.1, 0.01, and c.c. inocula were 23, 29 and 26 per cent for the R39 tumor; 13, 13 and a per cent for the Flexner-Jobling tumor; and 0, 3.2 and 3.8 'per cent for the 256B tumor. Statistical analysis showed that the variations in the percentage of regressions are not significant 8 The percentage of regressions was obtained by dividing 100 times the number of regressive tumors by the sum of regressive and progressive tumors. A nodule that attained a mean diameter of 5 mm. or more and disappeared was considered a regressive tumor.

6 TRANSPLANTABLE RAT TUMORS 369 (P = 0.27 to 0.75). The data, then, indicate that the size of the inoculum does not have any perceptible influence on the percentage of regressions. Another method of measuring the immune response of the rats to the R39 sarcoma is the use of the inhibition constant (8). Table V gives a statistical TABLE V: Effect of Size of Inoculum on the AlJerage Inhibition Constants of RJ9 Sarcoma Average Inoculum No. of tumors inhibition Differences] constant 0.1 c.c, ± ,01 c.c, O± c.c, ± ± 57% ±161 % Probable error. significant. t Differences with probable errors greater than 33 per cent are not analysis of the effect of size of inoculum on the average inhibition constant. The average constant of the tumors developing from 0.1 c.c. inocula was ; from 0.01 c.c., was ; and from c.c., was The differences in the average inhibition constants were not significant. An additional statistical test was made to determine any possible relationship between the inoculum and the inhibition constant. A contingency table was prepared with these factors, and Xl and P were calculated. P was found to be 0.78, which indicates that there is no' association between the size of the inoculum and the inhibition constant. Statistical analysis indicates, then, that the size of the inoculum has no perceptible effect on the inhibition constant. DISCUSSION The size of the inoculum has been shown to have a pronounced effect on the percentage of takes and the latent period, and also a slight effect on the growth constant. Depending upon the size of the inoculum, the percentage of takes may vary from 0 to 100 per cent and the latent period may vary from zero to thirty-six days. If the size of the inoculum is not kept constant, variations in tumor growth are to be expected. The control of the size of inoculum in experimental work on transplantable tumors is therefore essential. Measurement of Susceptibility to Transplantable Tumors: It is well known that individual animals of the same species vary in their susceptibility to drugs, toxins and bacteria. Some individuals succumb to small amounts of these agents, while others require much larger quantities. The measurement of the susceptibility of an animal group necessitates the use of graduated inocula, and the injection of each inoculum into a large number of animals. The susceptibility of the group can then be represented by an S-shaped mortality curve (10). The present experiments show that animals vary similarly in their susceptibility to transplantable tumors. It has been observed that only 34 per cent of albino rats are susceptible to Walker tumor 256A when the rats are

7 370 ROBERT SCHREK inoculated with c.c. amounts of a suspension, whereas nearly all the rats (97 per cent) are susceptible when inoculated with 0.1 c.c. Even when homogeneous strains of animals are used, the individuals have different degrees of susceptibility, as can be seen from Shear's (6) and MacDowell's (5) work. MacDowell, using mouse strain C58, found that 10 per cent of the mice were susceptible when 300 leukemia cells were inoculated and that 100 per cent of the mice were susceptible when 312,000 cells were inoculated. MacDowell's data can be represented by a typical S-shaped mortality curve. With another mouse strain (89), he found that only 74 per cent of the mice succumbed to a standard inoculum of leukemia cells. Likewise, Cloudman (11) found that 31 per cent of a pure mouse strain (D) succumbed following the inoculation of an arbitrary amount of a transplantable carcinoma. Presumably, the percentages of susceptible mice of MacDowell's strain 89 and of Cloudman's strain D would have been different if they had used a different inoculum. In contrast to this quantitative conception of susceptibility, geneticists have assumed that animals can be classified into two distinct groups-susceptible and non-susceptible. It may be that hybrid animals do have qualitative differences in susceptibility, but individuals of homogeneous strains would be expected to differ only quantitatively. The measurement of the susceptibility of hybrid and pure strain animals by determining the percentage of animals that succumb to a standard arbitrary inoculum seems inadequate. The method does not establish the geneticists' assumption that animals can be classified into two distinct groups. SUMMARY Experiments on four transplantable rat tumors showed that the size of the inoculum has: (1) A marked effect on the percentage of takes; the smaller the inoculum, the less the percentage of takes. (2) A marked effect on the latent period; the smaller the inoculum, the greater the latent period. (3) A slight effect on the growth constant; the smaller the inoculum, the smaller the growth constant. (4) No perceptible effect on the percentage of regressions and the inhibition constant. BIBLIOGRAPHY 1. BASHFORD, E. F., MURRAY, ]. A., HAALAND, M., AND BOWEN, W. H.: General results of propagation of malignant new growths, Third Scientific Report Imperial Cancer Research Fund, 1908, KOENIGSFELD, H., AND PRAUSNITZ, C.: Zur Frage der Filtrierbarkeit transplantabler Miiusecarcinome, Zentralbl. f. Bakt. (Abt. 1) 74: 70, FRANKEL, ERNST: Untersuchungen uber die Roustumoren beim Huhn, Ztschr. f. Krebsforsch. 25: 407, COSTA, ANTONIO: Untersuchungen uber die zur Obertragung experimenteller Geschwillste notwendige Zellenzahl, unter Bezugnahme auf die Filtrierungsversuche und auf -die Pathogenese der Metastasen, Ztschr. f. Krebsforsch. 36: 399, MACDOWELL, E. C.: Genetic aspects of mouse leukemia, Am. J. Cancer 26: 85, 1936.

8 TRANSPLANTABLE RAT TUMORS SHEAR, M. ].: Chemical studies on tumor tissue. III. Titration of mouse tumors, Pub. Health Rep. 51: 668, SCHREE, ROBERT: A quantitative study of the growth of the Walker rat tumor and the Flexner-Jobling rat carcinoma, Am. J. Cancer 24: 807, SCHREE, ROBERT: Further quantitative methods for the study of transplantable tumors. The growth of R39 sarcoma and Brown-Pearce carcinoma, Am. ]. Cancer 28: 345, SCHREE, ROBERT: Permanent and transient (fortuitous) variations of the growth components of transplantable rat tumors, Am. ]. Cancer 28: 372, TREvAN, ]. W.: The error of determination of toxicity, Proc. Roy. Soc. London, ser. B 101: 483, CLOUDMAN, ARTHUR M.: A comparative study of transplantability of eight mammary gland tumors arising in inbred mice, Am. ]. Cancer 16: 568, 1932.