Chemotherapeutic Experiments on Rat Tumors1 Richard Weil, M.D

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CHEMOTHERAPEUTIC EXPERIMENTS ON RAT TUMORS1 RICHARD WEIL, M.D. From the Huntington Fund for Cancer Research and the Department of Experimental Medicine of the Cornell University Medical College, New York City (Submitted for publication, December 17, 1915) In discussing a subject of such extent as the chemotherapy of tumors it is quite evident that only isolated features of the problem can be touched on. In the present paper I shall attempt to discuss certain phases of the work which I have been carrying on for several years, in their bearing on the general problem. 1. The penetrability of the living tumor cells. There is at the present time considerable unanimity on the subject of the in- travitam staining of cells. Goldmann (3), who was one of the first to study the distribution in the cells and tissues of dyes which were introduced into the circulation, reached certain conclusions which have largely served as the point of departure for subsequent study. He found that certain of the cells took up these dyes, which could then be identified as characteristically colored granules distributed through the cells. Goldmann considered that these granules were preformed elements of cell structure, which had been stained by the dyes. It is possible that this explanation holds true of certain basic dyes (such as janus green, methylene blue, neutral red). Upon this theory it is clear that a staining of granules is simply an alteration of paraplasm, or deuteroplasm, and that, in the absence of a diffuse staining of the cytoplasm we cannot speak of a true vital stain of the cells. Indeed, Fischel (2) in Ehrlich’s Encyclopadie, holds that there is no such thing as a vital stain in the latter sense.

l Read in the Symposium on Cancer Research of the Second Pan-American Scientific Congress, Washington, D. C. , January 5, 1916. 95 96 RICHARD WEIL

On the other hand, it is now widely believed that staining of preformed granules is not the correct explanation in the case of the dyes used by Goldman, which belong almost entirely to the beneidine group. Evans and Schulemann (1) have argued quite convincingly that the presence of the intracellular granules in the cells, in the case of this group of dyes, is due solely to phago- cytosis. The cells take the circulating particles of dye out of the circulation, and they then appear as densely stained particles within the cell body. In harmony with this view, it is found that only certain definite types of cells, which act as phago6ytes- the “scavenger cells” as Evans calls them, can take up these dyes. The living cells of tumors do not belong to this category, hence it is impossible to stain them in vivo with the bemidine dyes. Indeed, I have not been able to stain them with any dyes whatever. The cause of this differentiation between cells is probably biological rather than physical. The scavenger cells are differentiated to pick foreign substances out of the blood, for the purpose of elimination. They are, so to speak, a widely disseminated excretory organ. This view is borne out by some instructive experiments carried out by Kite (6). He found that certain dyes (such as azolitmin, congo red, tropeolin 000 No. 1, alizarin sulpbonate, indigo-carmin) which fail to penetrate amoeba proteus, diffuse freely throughout the organism when injected into it. In other words, the surface of the cell offers an obstacle to its entrance; once in, the color is taken up diffusely by the protoplasm. Again, he states that “if the egg of Asterias be punctured, the acid dyes used penetrate the swollen area for varying depths, but never enter the normal unswollen protoplasm.” Consequently, it is found that dead or injured cells behave quite differently towards the beneidine .dyes. Their peripheral resistance is gone, and they take up the dyes rapidly, presenting a uniform stain. Thus the cells of the kidneys of rabbits treated with sublimate or cantharidin (Gross (4)), and the anterior horn cells in experimental poliomyelitis (MacCurdy and Evans (7)) may be strikingly stained by these dyes. In this connection, the claims of Wassermann and Keysser (10) CHEMOTHERAPEUTIC EXPERIMENTS ON RAT TUMORS 97 with reference to the staining of living tumor cells, are worthy of attention. They asserted that the eosin penetrates all the cells of the body, and therefore used it as a carrier (“cytotrochin”) for selenium. Their facts, however, do not bear out this asser- tion. They never succeeded in staining tumors smaller than a cherry pit in size-in other words, tumors in which central necrosis had not occurred. Benign spontaneous tumors, which have no tendency to undergo necrosis, they invariably failed to stain. Internal implantations, which have an infiltrative mode of growth and are well supplied with vessels, Keysser (5) was never able to stain. It seems quite clear, therefore, that they did not succeed, as they thought, in staining the living cells, but only the necrotic areas. In a recent paper (11) I have critically analyzed these results. 2. Staining of necrotic areas. Inasmuch, therefore, as the conditions do not permit of staining the living cells of the tumor, one is perforce driven to a study of the staining reactions of the necrotic parts of tumors. It is this phase of the problem to which I have chiefly devoted my attention. The literature which bears on the subject is to be found almost exclusivelyin the remarkable series of studies which have been published in the last few years from the Sprague Memorial Institute (8) under the direction of H. G. Wells, dealing very largely with the staining reactions of tuberculous tissue. The first question which was studied in my work concerns the distribution of crystalline substances in necrotic tumors, as compared with the normal tissues of the body. Rats bearing necrotic tumors received intravenous or subcutaneous injections of solutions of sodium iodide. After varying intervals of time the animals were killed, and the various tissues of the body were analyzed for their iodine content. The chemical work was carried on by Dr. Van Alstyne, to whom my thanks are due. It was found that the blood regularly contained the largest proportion, and after this came the tumors and the liver; the other tissues, except the kidney, have regularly shown very much less iodine. If the tumor was small and non-necrotic, its proportion of iodine was very small. These findings are entirely in harmony 98 RICHARD WEIL with those recently published by Wells, DeWitt and Corper (12) on the distribution of potassium iodide in tuberculous tissue. They interpret their results as indicating that “the large amount of iodine present in necrotic tissues, whether tuberculous or otherwise, is dependent on purely physical conditions, i.e., the destruction of the semi-penetrability of the cells.” 3. Localization of dyes in tumors. The relation of the dyes to the necrotic tissues of tumors is of considerable interest. Wells, DeWitt and Corper arrived at the conclusion that “necrotic tissues, whether tubercles or other lesions, behave like any non-living colloidal mass into and from which crystalloids diffuse readily and rapidly, while colloids enter very slowly or not at all.” In support of this view they showed, by a very ingenious application of anaphylactic methods, that egg white does not penetrate the caseous tubercle. However, their theory does not appear to take account of all the facts. Thus Dr. DeWitt (13) herself has shown in another paper that caseous tubercles can be thoroughly penetrated and stained by trypan red and by trypan blue. Both of these dyes, however, are colloidal. As an actual fact, the relationship between foreign colloids in the circulation and necrotic tissues are very much more complex than might be inferred from the hypothesis above outlined. In illus- tration of this fact I might instance the following observation. During the period in which I was studying the distribution of dyes in tumors, the rats in our laboratory fell a prey to a very serious epidemic. The disease manifested itself as a pro- gressive caseating inflammation of the lungs. Macroscopically and microscopically the lesions presented a striking resemblance to human phthisis, without, however, showing cavity formation. Very frequently I autopsied animals in which not only necrotic tumor tissue, but also these caseous areas in the lungs were present. If such animals had previously been injected intravenously with appropriate dyes of the benzidine series, it was almost invariably possible to make a very striking observation. The dye stuff, e.g., congo red, regularly produced intense discoloration of the necrotic tissue in the tumors, but in no instance, over dozens of observations, did it produce the slightest staining CHEMOTHERAPEUTIC EXPERIMENTS ON RAT TUMORS 99 of the caseous areas in the lungs. Congo red, it may be added, is a highly colloidal dye. This statement is based on the fact that the dye does not diffusethrough membranes. Certain other properties of its solutions seem, however, to range it among the highly dissociated electrolytes. Such, for instance, are the effects upon the boiling and freezing points, and the fact that its solutions are optically clear. Therefore, one may conclude that the relation of colloids, or of colloidal dyes at least, to necrotic tissues is not uniform. As a matter of fact, the diffusibility of dyes into a colloidal mass depends upon a variety of circumstances. As regards the dye, not only its degree of diffusibility through animal membranes which determines its value as a colloid, but its electrical charge, its chemical reaction, and its chemical composition all play a r81e. As regards the colloidal mass again, its physical composition is of importance, as is also its electrical charge and its chemical composition. A knowledge of these factors permits of a fair guess, but no more, as to the result. Thus Teague and Buxton (9) found that agar, which is supposed to carry a nega- tive charge, was easily penetrated by acid dyes, anilin blue diffusing as actively as the much more slightly colloidal eosin. Of the basic dyes, only the slightly colloidal ones diffused in any amount. Even moderately colloidal basic dyes showed little capacity to invade the agar. The basic dyes, however, stain the agar intensely, whereas the acid dyes leave it uncolored. Congo red and azo blue constitute an exception to the latter rule, and although they are acid dyes, diffuse only slightly but stain intensely. Thus it will be seen that no generally valid law for the diffusion of all colloids, or colloidal dyes, into necrotic tissues can be formulated. The same dye may react quite differently to different types of necrosis even in the same individual. 4. Localization of the benxidine dyes. Of the benzidine group, a considerable number of dyes have been tested, starting with congo red. In all over 20 dyes have been included in the present study, all of which are well known chemical individuals of the benzidine group, representing substitutions either in the ben- zene or in the naphthalene nucleus by sulphuric acid, hydroxyl, 100 RICHARD WEIL methyl, salicylic acid, and other groups. In general these dyes tend to localize first in the peripheral zone of the central necrotic area. If repeated injections are made, they gradually penetrate the entire necrotic mass. Eventually, with the use of very large amounts, some of these dyes may slightly discolor even the healthy, actively growing rim of tumor tissue, owing to the presence therein of minute foci of necrosis. For the other tissues of the body, the stains have a varying degree of affinity, depend- ing on the degree to which they are taken up by the scavenger cells. Superficially it would appear that certain dyes have a relatively specific tropism, if one may use this term, for the necrotic areas of the tumor. It is, however, not safe to judge of the localization of the dyes by the discoloration of the skin. Animals which have been treated by intravenous injections may on autopsy present no apparent evidence of discoloration, either in the skin or in the internal tissues. If, however, the liver be boiled for a few minutes--a procedure long ago suggested by Ehrlich-the masked discoloration at once becomes evident. Observing this precaution, I have never yet found discoloration in the tumor which was not accompanied by some discoloration in the liver, either alone or with other of the parenchymatous viscera. On the other hand, many of the dyes appear to lodge in far greater amount in the necrotic area of the tumor than in any other tissue, and also to remain there for a longer period. Whether they are actually present in greater amount there than in the liver is, however, very doubtful, owing to the fact that the underlying colors make an ocular comparison extremely falla- cious. In some experiments, equal weights of liver and of necrotic tissues were suspended in equal volumes of water, and the colors of the resulting solutions were compared. It did not appear that the liver contained less than the tumors in any instance; indeed, it occasionally contained more. Thus, it is probably correct to say that none of the benzidine dyes mani- fests more than a relative specificity for the necrotic tumors. And it is not unlikely that even this apparent specificity is actually nothing more than the expression of a retarded rate of absorption from these poorly vascularized areas. It is interest- CHEMOTHERAPEUTIC EXPERIMENTS ON RAT TUMORS 101

ing to note that these dyes manifest the same predilection for the necrotic areas of human tumors. A solution of congo red was injected intravenously into a patient with cancer of the breast in the hope of helping in the detection of the carcinomatous areas at the subsequent operation. When the breast, with the axillary contents, was removed, it was found that the necrotic areas of the tumor foci, both in the breast and in the axillary nodes, were stained an intense red. The living areas of tumor tissue, as well as the normal tissues of the breast, appeared to have their normal coloration. It seems unlikely, however, that this method will ever prove of any clinical value. 5. Metachromasia. An .interesting phenomenon is the changes of color undergone by some dyes after they enter the necrotic areas. This color change has been described as metachromasia, adopting the terminology of pathologists. The cause of the phenomenon is somewhat obscure. It is, of course well known that some of the benzidine dyes are markedly affected in color by the mineral acids. Indeed, congo red has on this account been adopted as an acid indicator. But the organic acids have this effect only in high concentration and in minor degree. More- over, the color changes are not similar to those induced by mineral acids, but resemble more the changes induced by the localization of dyes in amyloid. In seeking to determine the cause of these changes I was led to test the effect of solutions of various polypeptids on the benzidine dyes. I found that they effect changes quite similar to those produced in vivo by the necrotic tissues. Thus if solutions of congo red be mixed with solutions of various amino-acids, a series of changes in color can be produced, slight, for example, in the case of glycyl-glycine, more marked with leucyl-asparagine and alanyl-glycine, and pronounced in the case of glycyl-glycyl-leucine. The color does not change to blue, as it does with mineral acids, but rather to a deep mahogany brown. Mr. Carruth has suggested to me that color changes in dyes like congo are to be explained by a separation of the base (Na) from the acid radical, which makes it possible for the dye to assume the isomeric quinone form. Al- though the free congo acid may exist in a red form (azoid), this 102 RICHARD WEIL form is not stable in aqueous solutions and passes instantane- ously into the blue form (quinoneimide). Intermediate shades- brown-violet, etc.-must represent the presence of certain amounts of each form:

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SOsH (Na) so3 At all events, it appears that the chemical conditions present in the necrotic tissues results in the production of some change in the character of the dye compound. In the same animal the normal tissues, such as the skin, which take up the dye, present it in an unaltered form. This fact led to the hope that the necrotic areas might conceivably be competent to break up compounds of these dyes with toxic substances, freeing the latter for attack upon the surrounding healthy tumor cells. 6. Therapeutic e$ects of certain compounds. The method of study followed in determining the effect of the dyes upon tumor growth, and a typical result, are illustrated by the following protocol. In this case the dye used was Columbia violet R, supplied by the Berlin Aniline Works. It is a diphenyl-disazo- di-amino-naphthol-sulphonic acid. A rat, series A, X, weighing 170 grams, has a sarcoma, the Buffalo strain, inoculated 27 days previously, now measuring $ by l+ inches, firm, not ulcerated. April 24, subcutaneous injection: 3 cc. 0.5 per cent C. v. H. No constitutional efYects ; no discoloration except at site of injection. April 25, the same dose. April 26, the same dose. April 27, no general discoloration. Exploratoiy section of tumor: center necrotic, peripheral necrotic rim shows a violet discoloration. Two pieces removed from healthy margin and planted in 10 rats, in all of which “takes” occurred. Skin sewed up and soon healed. May 11, 1; cc. 1 per cent C. v. R., intravenously. Tumor has grown to 1 by 2+ inches. CHEMOTHERAPEUTIC EXPERIMENTS ON RAT TUMORS 103

May 13 and 14, 2 cc. 1 per cent, intravenously. May 17, animal died. Autopsy: Tumor unstained in growing margin. Necrotic core shows all transitions in color from salmon yellow at center to violet at margin. Infiltration of lungs, with severe caseation. No discoloration. Liver and kidney on boiling show slight discoloration. A series of compounds analogous to congo red were made for me by Mr. Carruth, working under Professor Orndorff in Ithaca. In determining the compound to be made we were perforce guided by largely speculative considerations as to their probable effects upon the living cells. Unfortunately the data upon which such calculations can be based are few and inadequate. Aside from this consideration, however, it is evident that chemotherapeutic compounds, to be of any possible service in the treatment of tumors, must possess certain other properties. They should not be highly toxic to the organism. They must, of course, be soluble. They should be fairly stable in solution, yet should be dissociable in the necrotic areas of the tumors. These properties are not such as can be foretold of any given compound with certainty, hence the investigation resolved itself into an empirical study of such of the compounds as seemed most favorable. The compounds which were tested. out on the rat tumors are comprised in the following list:

In solution 1. Ortho-diselenide dye. 2. Congo-formaldehyde compound. No free formaldehyde present. 10 grams congo red per liter; 0.43 grams of formaldehyde per liter. 3. P’-arseno-aniline dye 1 per cent. 4. Mercury congo blue, 0.5 per cent; probably a mercuramino compound. In powder form 5. Recrystallized congo red. 6. Recrystallized bordeau extra. 7. Barium salt of congo red. 104 RICHARD WEIL

8. Potassium salt of congo red. 9. Zinc salt of congo red. 10. Copper salt of congo red. 11. Copper salt of bordeau extra. 12. Congo di-triazine.

14. Thio-aniline dye.

16. BenzidineGa:ghionic acid. 17. Soluble selenium congo red. 18. Salmon red, thiazol derivative. The exact composition and probable formulae of these compounds will not be here discussed. Consideration of solubility, toxicity, and other properties will also be deferred to a future publication. The present paper is concerned only with the analysis of their therapeutic effectiveness. All the compounds appeared, judging by the gross discoloration of the tissues, to localize electively in the necrotic areas of the tumors. All the injected animals in which tests were made, however, showed discoloration of the boiled livers. The localization was further controlled by chemical analysis of the organs which, at least in the case of the arsenic compound, could be considered to give reasonably accurate results. The maximum yield of arsenic per gram of substance was obtained from the liver, while the tumor and the kidney came next. The arsenic content of the other organs and tissues was low. From these results it would appear that localization in the tumors was only relatively specific. It is, however, possible that the arsenic reached the liver only after the compound had been dissociated in the necrotic areas of the tumors. In judging of therapeutic effects, three criteria were employed, namely, the rate of growth of the tumors, their transplantability, and the number of retrogressions. The details of the method have already been illustrated. None of these three criteria has CHEMOTHERAPEUTIC EXPERIMENTS ON RAT TUMORS 105 an absolute value in the type of tumor which formed the basis of this study, inasmuch as they all vary to a remarkable degree. ,4t times the tumor “takes” in a large percentage of the inocula- tions, while at other times, for no ascertainable reason, this percentage is greatly reduced. The rate of growth and the percentage of retrogressions also vary strikingly in different gen- erations of the tumor. For this reason it is always necessary to plant a considerable series, of which at least half are kept as controls, while the remainder are reserved for the purposes of the experiment. The conditions are unfortunately such as to preclude the determination of small effects; on the other hand a definite and considerable influence on the life history of the tumors could certainly not escape detection. Judged by these stand- ards, the results obtained were not encouraging. In only three out of the entire list, namely, numbers three, four and fourteen, were any effects ascertained, and these three proved so highly toxic to the rats when given in therapeutic amounts that it seemed questionable whether the effects on the tumors were attributable to the specific action of the drug, or to the general effect upon the health of the animal. At all events, none of the substances possessed that combination of properties which would make them available for the effective treatment of the rat tumor. The principal object of the investigation, therefore, failed to be accomplished. I wish to acknowledge the constant help and advice of Pro- fessor Orndorff and of Mr. F. E. Carrutli, without which this work would have been impossible.

CONCLUSIONS 1. Living tumor cells are not penetrated by colloidal dyes. 2. The necrotic areas of tumors contain a larger amount of iodine than do the other tissues of the body after the intravenous injection of sodium iodide. 3. The necrotic areas of tumors present an intense discoloration after the intravenous or subcutaneous administration of dyes of the disazo group. 106 RICHARD WEIL

4. The discoloration of these tumor areas is very frequently associated with some discoloration of the liver, while the other tissues of the body remain macroscopically unstained. 5. The staining of the necrotic areas of tumors is not due solely to the death of the cells, inasmuch as areas of pulmonary caseation in the same rats do not present any discoloration. 6. The localization of colloidal dyes in necrotic tissues is not a simple physical phenomenon, subject to the laws of diffusion of fluids into non-living colloidal material. The diffusibility of the dyes through membranes, as also the electrical charge, the chemical reaction, and the chemical composition of both colloids influence the result. 7. A peculiar alteration in the color of dyes of the benzidine group occurs in necrotic areas. This has been described as met achromasia. 8. A series of new synthetic compounds analogous to congo red were injected into tumor bearing rats. No definitethera- peutic effect could be determined.

BIBLIOGItAPIlY

(1) EVANSAND SCHULEMANN:Science, 1914, xxxix, 443. (2) FISCHEL: Ehrlich's Encyclopadie Mikroskopischen Technik, Urban and Schwarzenberg, Berlin, 1903, i, 349. (3) GOLDMANN:Beitr. z. Klin. Chir., 1908, lxiv, 192. (4) GROSS:Beitr. z. path. Anat., etc., 1911, li, 528. (5) KEYSSER:Ztschr. f. Chemotherap., 1914, Orig., ii, 188. (6) KITE: Biol. Bulletin, 1913, xxv, 1. (7) MACCURDYAND EVANS:Berl. Klin. Wchnschr., 1912, xlix2, 1695. (8) Studies from the Sprague Memorial Institute, Chicago, 1914, vol. ii. (9) TEAGUEAND BUXTON:Ztschr. f. physikal. Chem., 1907, lx, 464. (10) WASSERMANN,KEYSSER AND WASSERMANN: Deutsch. med. Wchnschr., 1911, xxxvii, 2389; Berl. klin. Wchnschr., 1912, xlix, 4; Ztschr. f. Chemo- therap.,l914, Orig., ii, 110. (11) WEIL: Jour. Am. Med. Assn., 1915, xliv, 1283. (12) WELLS,DEWITT AND CORPER:Ztschr. f. Chemotherap., 1914, Orig., ii, 110. (13) DEWITT: Jour. Infect. Dis., 1914, xiv, 498.