ON THE APPARENT VARIATIONS IN THE RATE O F GROWTH IN GRAFTABLE MOUSE CANCERS?

By CHARLES WALKER, I).Sc., M.R.C.S., L.R.C.P., and HAROLD WHITTINGHAM, M.B., CH.B.

FTOnk thc Research Deparlmnt, Royal cancez Hospital, Glasgoio.

SEVERAL observers have recorded alternations or waves in the rate of growth in transmissible mouse cancers. Thepe variations in the rate of growth involve many generations of tumours, but do not show any real regularity in alternation between rapid and slow growth. For many weeks the tumours grafted into consecutive batches of mice grow more and more quickly. The rapidity of growth eventually reaches a maximum, diminishes, apparently reaches a minimum, and then increases again. These alternations follow upon each other as long as the observations have been continued in any series of experiments.

Bashford, Murray, and Bowen (1906 l) interpret their observations upon this point as demonstrating the existence of a rhythm in the growth energy of the tumours with which they have worked. They believe that the intrinsic power of multiplication and vitality of cells, derived from a primary malignant growth, vary in the subsequent generations of cells, and thus produce varia- tions in the rate of the growth of the tumours produced by grafting, each variation being possibly extended over many tumour generations, that is, through many consecutive inoculations of different batches of mice.

Calkins (1908 a), in reporting similar waves of growth, concludes that they are brought about by some cause within the cancer cell itself, and considers that this cause is almost certainly an intracellular parasite similar to Plamwtlio- phora braasice.

Ehrlich and Apolant (19059, 19064), in working with graftable mouse cancers, found that successive sojourns in fresh individuals as hosts, if the hosts were of the same near ancestry, increased the percentage of successful graft- ing. They also found that very rapid passage through successive hosts increased the rapidity of the growth of the tumours to an enormous extent. Ehrlicli and Apolant state that they were carrying out a definite plan in using a great number of animals and grafting as rapidly as possible. They say : ‘‘ Our object was to increase the malignancy of the tumour cells to the maximum by the continued systematic passage from animal to animal according to thc analogy of bacteriological technique.’’

Received July 26, 1912.

RATE OF GROWTH ZN MOUSE CANCERS. 233

The experiments described here are practically a repetition of sonie experiments done four years ago by one of us in the Cancer Besearch Laboratories in the University of Liverpool. The results of these experiments were not published. The methods and details in the present series of experiments are more satisfactory and complete.

It is common knowledge that no two living organisms are alike, that no two similar parts of similar organisms are identical, and that this difference extends to cells, for no two cells are morphologically identical. But the differences between cells extend beyond their morphological characters, for every cell differs from its neighbours, even if only by a very little, in its power of resistance to unfavourable factors in the environment, of response to various stimuli, and in its potentiality for growth and multiplication. Moreover, as existing cells vary from each other, so the cells produced by division must vary from each other, and from the cells that produced them. In these inoculation experiments with mouse cancer we have therefore two outstanding sets of varying potentialities : those of the individual mice into which the tumour cells are introduced, and those of the cells themselves. Now, theoretically, it should be possible to select particular and obvious characters in either the hosts or in the tumour cells.

In the experiments here described the tumour cells were selected in regard to the character of rapidity of growth. The procedure was as follows:-

Twenty mice were grafted at the same time from a well-established tumour, removed from a mouse of a ditferent breed. The tumour had been carried on for five years in mice bred in &sex. It was grafted into mice bred at Langside, near Glasgow. When two or three of the tumours produced from the grafts were large enough, twenty more mice were grafted from the largest one among them. This was carried out with five successive batches of mice, the largest tumour being chosen for grafting in each case. On the other hand, one of the most slowly growing tumours was chosen at a later date from one of the original batch of mice, and was used to graft another twenty, and 80 on, selecting the most slowly growing tumour. The results are shown in the accompanying figure (p. 234), which gives the average sue of the tumours in each batch of inice in consecutive weeks. The differences in the rate of growth are so striking as to require but little comment. In the vertical line the measurements of the tumours in square millimetres are given. These were obtained by multiplying together the two longest diameters of the tumours. This, of course, gives but a rough estimate of the differences in the sizes of the tumours, but rather diminishes than increases the real differences between them. We give our reasons for being satisfied with this rough estimate later. Throughout the experiments we have used small pieces of tumour for grafting, making them as nearly of a size as possible, and taking them from the healthy growing periphery. We have avoided the use of emulsions of tumour cells, though this method has been adopted by many observers.

Bashford and others (1 9 0 8 s, have emphaeised the necessity for using accurate doses of tumour cells, stating that this can be assured

D

WEEKS FROM DATE OF GRAFTING. - A. &tch grawon Oecf 4M /9/L (0ng;nd batch) B. .. ., .. *, 276 ,, (fmm one o f original batch) c. ,, ,, Jah26?/9/i? (from one o f &&h S;, D. .. .. ., h6? /O? ., (fm one of batch C!I

-x-x- E. ., ., .. .. 23!? .. (from one o f bard 0) -0-0- F . .. ,, .. ,, 2+% ,, (from one o f ong/na/batch) - G. ., I ,, May 69 .. (fm one o f &tchlF/

- --_- -.-.-

RAT8 OF GRO WTU IN MOUSE CANCERS. 235

only by using emulsions, and that only thus can certain errors be eliminated. However desirable accnracy of dosage may be, we con- sider that it cannot be gained by using emulsions of cells, as only living cells are effective. The variation in the amount of damage done in making emulsions in different instances more than counterbalances, in our opinion, any difference in the size of the solid portions of tumour introduce% Of course, in either case an unknown proportion of dead cells must be present.

In the measurements we give here we make no pretence to very great accuracy, which in our opinion would be quite unattain- able. The third dimension-that from the surface through the tumour towards the body of the animal-is entirely neglected, as it would in many cases be quite impossible to obtain it. We attach but little importance to the measurements made in the first week or fort- night after grafting, as so many sources of error exist until the tumour has reached a considerable size. This is emphasised by the fact that in some of the batches of mice the tumour growth war3 apparently more rapid during the first week or two than in other batches of mice where the rate of growth eventually proved to be many times as great. This was probably entirely due to inflammatory processes, and had nothing to do with the tumours themselves. I n the large tumours, however, the measurements are probably very nearly those of the actual tumour tissue, and in any case the differences are so great as to be beyond explanation as the result of chance. Also as the different rates of growth produced existed simultaneously, having been obtained from the same tumour in the same mouse at the same time, they cannot be due to any rhythm of growth energy in the tumour cells or in a parasite causing the malignant growth. We believe that the variations in the rate of growth can only be explained as the result of selecting those cells which varied towards rapid and slow multi- plication respectively. W e believe also that certain other phenomena connected with these graftable tumours are to be explained in the same way. When six years ago Professor Ehrlich gave a graftable mouse cancer to the Cancer Research Laboratories in Liverpool, it was found by one of us that a t first only about 30 per cent. of grafts in Essex mice were successful. After many successive inoculations, however, practi- cally 100 per cent. of successes were obtained. When this tumour was first used with mice obtained from Langside, many grafts failed to grow, but at present the percentage is practically 100. We secured a mouse bred in Langside with a primary vaginal carcinoma. After passing this through one batch of mice, we obtained practically 100 per cent. of successful grafting8 in Langside mice, and this has con- finued. But this tumour was paseed through several batches of Essex mice before as good a result was obtained.

We interpret these phenomena aa being due entirely to selection. The tuxnour cells vary among themselves in their power of resisting a

236 CHARLES WALKER AND HAROLD WUITTINGNAM.

strange environment. Only those possessing a high degree of resist- ance survive when a graft is introduced into an animal of a different breed from those in which the tumour has become well established. The offspring of these resistant :cells vary froin the cells from which they arise and from each other, some towards greater, some towards less resistance. Those varying favourably survive to divide and pro- duce fresh generations of cells, until a race of cells is established which is highly resistant to the environment, unfavourable variations being eliminated as they occur. If this race of cells is introduced into a fresh environment, the same process is gone through again if any of the cells are sufficiently resistant to survive and divide.

Selection would also appear to explain why Ehrlich and Apolant were able to produce very rapidly growing tumours by rapid trans- plantation through a number of animals. They necessarily selected the most rapidly growing cells in the process.

The great differences which exist between the behaviour of these mouse cancers produced by grafting and that of primary malignant growths seem to be to some extent explicable upon these lines. Most of those who have studied the subject believe that the peculiar characters of cancer cells are due to their having passed out of the control of some common influence (somatic co-ordination [Walker, 1906 cell autonomy [Ewing, 1908 9, or something of this nature which regulates the growth and the relations between the various cells and groups of cells forming the body ; that cancer cells, in fact, possess in some respects the characters of separate individuals living as parasites upon the organism. The process of grafting must involve selection of those cells most resistant to changes of environment, and must accent- uate in the succeeding cell generations the characters of independence. It is probable that thus such characters 88 the invading of surround- ing tissues are lost to a great extent, as they are not the subjects of selection. It must be realised that the same race of tumour cells, derived‘from the same primary growth, may be carried on by a pro- cess of grafting for a period of time exceeding many times the length of the normal life of the mouse.

REFERENCES.

1. UASHFORD, hfuRmY, AXD “Expcriniental Analysis of the Growth of BOWEN Cancer,” I’roc. Roy. Sot. London, 1906,

vol. Ixxviii. (B), p. 196. 2. CALKINS, GARY N. . . . “The So-called Ilhythm of Growth Energy in

Mouse Cancer,” Joz6rn. Exper. Meti., Ealti- more, 1908, vol. x. p. 283.

3. EHRLICH AND APOLANT. . “ Beobachtungen iiber maligue Maustuiii- oren,” Uerl. k h . Tchnaclir., 1905, Ed. xlii. 8. 671.

RATE OF GRO WTH m MOUSE CANCERS. 237

4. APOIAXT AND EHRLICH . “ Experimentelle Beitrage zur Geschwulst- lelire,” b’erl. klin. Wchnsclir., 1906, Ed. xliii. S. 37.

5. EASHFORD, I fURRAY, AND ‘ I Resistance and Susceptibility to Inoculated Cancer,” Third Scienta3c Report, InzpeTial Cancer Reseal-cli Fund, London, 1908, p. 262.

6. WALKER, CHARLES . , . ‘ I Essentials of Cytology,” Constable, London, 1907, p 7 ; Jozrrn. Path. and Bacteriol., Cambridge, vol. xvi. p. 185 ; Science Pw- gres6, vol. vi. 1912, April, p. 670.

5 . KWING, JAMES . . . . “ Caiicer Problems,” A ~ c h . Intern. Medicine, 1908, vol. i. p. 175.

HAALASD