OBSERVATIONS ON SOME GROWTH CHARACTERISTICS OF T H E WALKER CARCINOMA 256

S. MICHAELSON, D . v . M . , ~ AND J. A. ORCUTI., PH.D.-/-

ARLE in 1935 described the early history E and cytology of the Walker tumor. This tumor has been used extensively since 19373 in the study of tumor inhibitors. Experience with the Walker iumor by many investigators in various laboratories revealed that it pro- duced a uniiormly rapid growth, showed very little regression, and was readily adaptable to a variety of strains of rats.

Various methods lor administering a drug and evaluating its effect on a tumor have been utilized. These include single and multiple closes of the test material; gross and histological evaluation ot tumor damage;ly determination of the comparative weights of tumors in treatcd and untreated animals;5 measurement of the diameter of the tumor after a selected period of therapy;1° and comparison of tracings o f tu- mor si7e for evidence of reduction in siLe.1- 1*,11

In the investigation to be described, the Walker tunior was chosen because of the ease with which i t could be standardi7ed and re- produced. In order to maintain an adequate drug level, the agcnt wa5 incorporated in the diet at a concentration slightly lower than the previously dcterniined highest-tolerated dose. The incorporation of the test drug in the diet has been utilized lor several years and has been iound to be a satisfactory method for adminis- tering the agent.1- 6 8

The use of histological evidence or the use of comparative weights as an index ot damage to the tumor has the disadvantage of being feasible only after traumatizing the tumor by biopsy or excision. The adoption of a method that can be used periodically during the course of the experiment appears to offer the most reliable index of the ability of the drug to in-

From Eaton Laboratories, Yorwich, New York. The authors are indehted to Dr. C. Chester Stock and

Dr. K. Sugiura of the Sloan Kettering Institute for Can- cer Research, New York, New York, for offering many helpful suggestions and making availahle the Walker carcinoma 256 uscd in this study.

* Present addrcs: 1)epartmcnt of Radiation Biology, University of Rochester School of Medicinc and Den- Libtry, Rochester, New York t Present address: RikeI Laboratories, Inr , Los ..in-

geles, California. Received for publication, October 8, 195F.

‘l‘.4BLE 1 RATIO OF RATE OF GROWTH OF TUMORS

AMONG UNTREATED AND TREATED RATS _________ Concentration of . Slope ratio,

Drug drug iu diet, 7, treated/untreated _ _ _ _ _ ~ _ ~ ~ _ _ _ _ ~ _ _ _ {Trethane 0.60 0 60

0.50 0.36 0.25

0.62 0.75 0.85

0.22 0.83 0.13 0.51

Colchicine 0.002 0.81 0.001 0.94

Alloxan 0.20 1.05

hibit the growth of the tumor. In our experi- ments, estimation of the volume of the tumor at specified times atter implantation and a comparison of the growth rate5 of tuiriors in treated and control rats were utilized as an in- dex of inhibitory activity.

Various transformations of the growth curves of the tumor were made, and it as found that a plot 01 the logarithm of the tumor volume as the ordinate and the logarithm of the day of measurement as the abscissa yielded a straight line. I t became apparent that such linearity of growth rate for tumors would lend itself to simple quantitation by a comparison 01 slope ratios oi growth after the administra- tion of various compounds or different doses of the same compound (Table 1) .

R~ATE’KIALS AND MI.:THODS

The male albino rats used in this study were originally derived krom the Wistar strain and inbred since 1923 at Rainbridge, New York. Transplantation was performed aseptically. The tumor material lor transplantation was obtained from rats that had been inoculated fourteen days previously. Kats weighing 100 to 150 gm. were used as recipients. The donor rat was mesthetiTed wi th ether; the tumor was excised and placed in cold, sterile, normal sa- line. Sections ot tissue 1 mm. in thickness were cut from the viablc peripheral portions of the tumor. The tumor and cut sections were picked up in a 13-gauge trocar that had been pre- viously dipped in alcohol and flamed. The

416

s o . 2 GROWTH CHARACTERISTICS OF LvALKER TLMOR * h / l i C / l U e h O ? l h‘ 0 1 - C U t l 41 7 trocar was inserted subcutaneously into the right flank of the host rat and passed along the lateral abdominal and thoracic walls to the right axillary region, where the transplant was placed; the trocar was withdrawn while the transplant was held in place with the finger.

In transplanting fragments of tumor to sev- eral hosts, which were to be divided into groups undergoing different treatments, there was some delay between removal of the tumor and implantation of the sections into the host anirnal. The variability that might be induced by this factor was minimired by alternating the implantations among the animals in the var- ious groups.

af ter the fragments of tumor were im- planted, thc rats were assembled in groups of five, one group serving as tontrols. The rats received feed and water ad libitum. ‘The ani- iiials in the control group received the basic diet and those in the groups to be exposed to the drug received the- badc diet plus the test drug incorporated by mixing in a standard feed mixer. Previous experience in this labora- tory had shown that the percentage of drug in the diet times 1000 gives approximately the number of milligrams per kilogram of body weight per day of drug ingested. Rats on a 0.1 per ccnt medicated diet will, therefore, consume on the average 100 ing. of drug per Kg. body weight per day.

On the seventh, eighth, ninth, eleventh, and fourteenth days after implantation, each rat was weighed and the tumor measured for length, breadth, and thickness with a pair of vernier calipers. Because of the spherelike na- ture 01 the tumor, its volume can be estimated readily from these three dimensions, according to the following formula:

length x hreadth X thicknea 0.56

derived from the formula for the volume of a sphere (4/3 x r3).

The mean of the logarithms of the tumor volume in cubic centimeters from each rat in a group was plotted against the log of the day of measurement, and a straight line was fitted to the points. Difference of tumor growth be- tween control and treated rats was noted by the change in slope and position of the curve.

-_ ~ - ~ _

RESULTS

Twenty-three serial transplants of the Walker tumor were perforined according to

DAYS AFTER IMPLANTATION

FIG. 1. Growth of Walker carcinoma 256.

the procedure outlined. It w a ~ found that this tumor adapted itself very well LO the strain of rat used. Of 800 rats subjected to this treat- ment, there was a 90 per cent incidence of “takes,” no regression, and a negligible inci- dence of infected tumors.

When the mean log of the size of the tumor was plotted against thc log of the day of meas- urement, the points invariably fell in a straight line with a slope that was consistent from series to series. I n twenty-three serial transplants, the rate oT tumor growth for control rats displayed a slope of 5.2714 with a standard deviation 01 0.8439. ‘l‘he method of fitting straight lines to the logarithins of the size of the tumor was tested by Arthur Dutton, Statistics Section, De- partment of Radiation Biology, University of Rochester School of Medicine and Dentistry, and found to be satisfactory for sumrnari7ing thc data. In order to determine what effect a drug with a known inhibitory effect would have on the linear growth rate ok the IValker tumor, urethane4 was used at various dowge levels as a standard in several of these series. Evidence of inhibition of the growth of the tu- mor was indicated by a smaller mean slope for the size of the tumor among treated rats than among concurrendy tested untreated rats. The curves of the growth rate for the tumors of treated rats did not show any departure from

418 CANCER Marcli-April 1957 Vol. 10

linearity according to the chi-square determina- tion. An example of this response i5 indicated in Fig. 1 .The logarithm of the volume 01 the tu- mor was plotted against the logarithm of the day of measurement. A straight line was fitted to the points for the untreated (control) rats and for the rats maintained on the urethane dict. T n the untreated group, each point rep- resents the mean volume for lour tumors, as one rat died bekore the fourteenth day. In the treated group each point represents the mean volume lor five tumors.

That straight lines could be fitted to these points is indicated by the chi-square deter- mination, which is 0.0216 for the untreated tu- mors and 0.0618 for the trcated tumors. The mean slopes of these lines ale 4.539 and 2.904 for the untreated and treated tuiriors respec- tively. In this particular experiment, the dif- ference in sise of tumors between the treated and untreated rats was significant for odd5 of 19 in 20 by the t test, starting on the eleventh day. The ratios of the mean slope of the rate of growth of the tumors in untreated lats com- pared with rats that received urethane, colchi- cine, or alloxan are listed in Table 1.

From these data it can bc seen that the ac- tivity of urethane in inhibiting the growth of the tumor is dependent upon the concentra- tion of the drug in the diet. In all experiments in which tumor-bearing rats were treatcd with urethane, there was a lower rate of tumor growth as compared with untreated tumor- bearing rats. In two experiments in which col- chicine waq used, there was also an inhibition 01 growth of the tumor. Alloxan caused a

slight enhancement of the growth of the tumor.

CONCLIJSION

The comparison of calculated slopc ratios for the growth of the Walker carcinoma 256 in treated and untreated rats receiving sub- cutaneous implants 01 tumor fragments can be used as an index of tumor-growth inhibition. This technique can be used for the screening of new drugs. The relative therapeutic index for new drugs can be obtained by comparing the mean slope ratio for the growth of the tu- mor in rats receiving these drugs with that in rats receiving standard preparations with known tumor-inhibiting activity.

SUMMARY

Some characteri5tics of the growth of the Walker carcinoma 256 have been described. Upon subcutaneous implantation in albino rats originally derived from the IVistar strain, there was a 90 per cent incidence o i “takes” and no regression. When the logarithm of the size of the tumor was plotted against the log- arithm of the day of measurement, the points invariably fell in a straight line with a mean slope of 5.2714 f 0.8439.

Incorporation of tumor-inhibiting drugs, such as urethane or colchicine, in the diet of the rat induced a smaller mean slope for tumor growth, with no significant departure from linearity. Tt is suggested that this technique can be used €or the screening of drugs that can suppress the growth ol tumors.

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