THE AMERICAN JOURNAL OF CANCER A Continuation of The Journal of Cancer Research

- - VOLUMEXXX AUGUST,1937 NUMBER4

THE TRANSMISSIBLE AGENT OF THE ROUS CHICKEN SARCOhIA NO. 1

JAMES W. JOBLING, M.D., E. E. SPROUL, M.D., AND SUE STEVENS, M.A. (From the Department of Patlzology, College of and Surgeons, )

That animal tumors can be successfully transmitted within the species has been known for many years. The process was easily understood so long as living cells were required for transmission, the growth presumably resulting from continued division of the viable tumor cells. The discovery by Rous in 19 11 ( 1) that a chicken sarcoma could be produced in normal fowl by in- jection of a cell-free extract of the tumor considerably altered the general point of view, but at the same time offered the hope that the nature of the responsible agent would eventually be established. The hypotheses which have stimulated widely varying experimentation may be roughly divided into those regarding the agent as of extrinsic origin and those seeking its source in the metabolic processes of the host itself. Interpretation of the development of the Rous sarcoma as the result of infection with a living parasite, a virus more related to the causative agent of chicken pox, vaccinia, herpes, etc., is supported chiefly by analogy. An- drewes (2) has been most impressed by the properties borne in common by the Rous sarcoma and the more readily accepted virus infections. Nor does he accept the variation in type of response as a serious discrepancy, since he shows that the viruses can stimulate hyperplasia as well as necrosis and that all gradations between the two can be found. This view is also held by Rous (3), who considers that his more recent work with the Shope papilloma has provided him with confirmatory evidence. A somewhat more tangible means of investigation has been supplied by the method of ultra-centrifugation. Ledingham and Gye (4) report the concentration, by this procedure, of "elementary bodies" from the Rous sarcoma filtrate which they believe represent the causative factor of the disease.

1 This investigation has been aided by a grant from the Josiah Macy, Jr. Foundation. 667 668 JAMES W. JOBLING,E. E. SPROUL AND SUE STEVENS Among the extrinsic causes of cancer we must also classify the irritants and perhaps, more specifically, traumatic effects. Wells (5) stated in 1931 that " the trend of these results of recent cancer research is in support of the old view that cancer is usually the result of protracted stimulation of tissue growth by non-specific agents acting on tissues, the susceptibility of which is deter- mined by their hereditary background." Knox (6) and Ewing (7) find no adequate support of a relationship between trauma and tumor growth, but the theory of a causative relation is still considered plausible by many (8). Re- cent stimulus has been offered by Wolbach's histological studies on the tissues acted upon by several synthetic carcinogenic substance^.^ He finds necrosis followed by normal repair which imperceptibly becomes transformed into a new growth. The group believing that the causative agent of cancer, or more specifically of the chicken sarcoma, is endogenous is further divided by diverse views as to its nature. It has been called an enzyme, without further elucidation of its actual nature (9). It has been likened to the hormones (10, 11) in be- havior and chemical composition, and there are those who believe more con- cretely that it may be derived from the sex hormones by some alteration in (12). Murphy (13) favors the term " transmissible mutagen" for the substance appearing in the cell-free tumor filtrates, indi- cating that the agent is capable of directing the specific development of the cell into abnormal channels and that it is further elaborated by this altered cell. Ludford (14) has also expressed somewhat similar ideas, although not stressing the transmissibility of the mutagen. A decade ago it seemed that Gye (15) might reconcile these opposing forces by his suggestion that an exogenous universal cancer agent, which he termed a virus, received its specificity when combined with an intrinsic factor, presumably elaborated by the various cell types. His experiments, however, were not corroborated by others nor by later developments in his own laboratory. Such unrelated views have provided the means of unearthing a formidable number of facts regarding the character of tumor growth without arriving at a tenable explanation of the course of events. This investigation was begun following the observation that independent studies on distinct phases of the problem indicated the importance of the lipids in abnormal and physiological growth processes. The most outstanding was undoubtedly the synthesis by Cook, Barry and their associates (16, 17) of a series of carcinogenic hydro- carbons in the form of condensed ring compounds, recalling the structure of . It would seem unlikely, in view of the prolonged period of ap- plication required for tumor formation, that the known chemicals act directly on the cells, but the remarkable consistency in carcinogenic activity observed in various laboratories leaves no doubt of the specificity of their action (18, 19). The significant changes which each compound suffers in the process of cell metabolism remain for future elucidation. The second most arresting observation was the alliance of the sex hor- mones with cancer and the timely discovery of the molecular structure of 2Reported at the Annual Meeting of the American Society of and Bacteriology, Chicago, March 1937. several of these complicated substances (19, 20, 21). The recognition by the biologists of the r6le enacted by the ovarian hormone in normal develop- ment and malignant growth of the mammary gland as well as in stiniulation of hyperplastic endometrial change in mice long preceded the isolation by the chemists of these hormones. Lathrop and Loeb (22) in 1916 demonstrated the reduction in the incidence of spontaneous tumors following castration or prevention of breeding and many have subsequently corroborated and ampli- fied this statement. A new type of investigation was offered by the discovery that the follicular and luteal hormones are also phenanthrene derivatives not unlike those stimulating the growth of tumors and structurally related to the (23). It remained then to demonstrate the actual production of tu- mors by injection of pure estrogenic compounds and this was accomplished by Lacassagne (24) working with a strain of mice known to develop spon- taneous mammary cancer. This achievement has been confirmed and the observation made by Loeb (20) that the estrogenic hormones can induce cancer only in those organs normally stimulated by them. Cook and Dodds (25) contributed to the subject by synthesis of a variety of compounds all possessing the tricyclic phenanthrene ring nucleus and biologically estrogenic as well as carcinogenic. The third group of studies, which led to the undertaking of our own experi- ments, had to do with the suggestion that lipids could be classified as growth- stimulating substances. Contradictory evidence has been presented by many authors whose methods of demonstrating the effect of increased lipid intake have differed widely. Robertson and Burnett (26) caused an acceleration of the growth of the Flexner-Jobling rat carcinoma by direct injection of choles- terol into the tumor; lecithin had the opposite effect. Subsequent publications by these authors as well as by Bernstein and Elias (27) and Moravek (28) have been in agreement in regard to the effect on this and other tumors of feeding and injecting these substances. One can also find the view that cholesterol, fed or injected, has no effect upon tumor development (29), and Harnes (30) claimed to have demon- strated a retarding effect when the inoculation of the Brown-Pearce carcinoma into the testis was preceded by subcutaneous administration of alcoholic brain extract in corn oil. Vassiliadis (31) embraces both assump- tions by the statement that stimulating and inhibiting substances can be found equally in the ether and acetone extracts of normal tissues tested against tar- induced tumors. The most recent observations on this subject are those of MacFadyen and Sturm (32), who demonstrated augmentation of growth of the Bashford mouse carcinoma No. 63 when the graft had been exposed to the ether-soluble fraction of mammary gland. The ether-insoluble material definitely interfered with tumor growth. It is hardly remarkable that there should exist such discrepancies when we consider that inhibiting and stimulating substances ordinarily form a close alliance, and variation in technic may easily favor the one or the other. At least the majority report some influence exerted by fats and related substances on tumor proliferation. The need for investigation of the activity of the lipid fraction of tumor tissue was further emphasized by numerous reports of the high lipid content 670 JAMES W. JOBLING,E. E. SPROUL AND SUE STEVENS of tumors as well as of the blood of tumor-bearing animals and patients. Bul- lock and Cramer (33) concluded from analytical studies on mouse and rat tumors that the phospholipids were associated with rapidity of growth. Sup- port for such a view can be found in the analysis conducted by Bierich and Lang (34), Yasuda and Bloor (35), and more recently by Haven (36). One also finds cholesterol stressed as an important factor in tumor metab- olism, reasoning from content alone. Roffo (37) has written extensively on the correlation of high cholesterol level with the growth of tumors, a view subscribed to by Correa (38) on the basis of analyses of tumors and the blood and non-tumor bearing organs of tumor animals, as well as the personal ob- servation that cholesterol, administered orally or by subcutaneous route, stim- ulated the growth of tumors. Moravek develops the subject still further when he states that (( cholesterol, while the cause of the chemical, physico- chemical and morphological differences between the tumor cell and a normal one, is a product that originates from some other substance. This substance is the first and direct cause of the origin of the tumors " (28). Whether such an emphatic statement can be upheld remains to be seen, but one cannot but be impressed by the number of workers who consistently found that tumor tissue possessed an excessive amount of fat and related substances (39, 40, 41, 42, 43). In regard to the Rous chicken sarcoma No. 1 in particular, the attempt has repeatedly been made to separate the active and inactive portions of the filtrate. The majority have looked for success among the less drastic proce- dures of the chemists, in recognition of the fact that the sarcoma agent, while hardier than that of most tumors, cannot withstand many ordinary manipula- tions. The adsorption phenomenon was particularly adaptable since a variety of inert substances could be employed without the application of heat, and following its use by Leitch (44) several investigators reported a partial puri- fication of the transmissible agent by this means. Lewis and Mendelsohn (45) first linked the agent with the globulin fraction of the tumor by pre- cipitation with half saturated ammonium sulphate and dialysis, concluding that the union might be a loose chemical one, a physical combination, or that the agent itself might be a globulin. Murphy (46) and his coworkers on the other hand separated the from the active principle by adsorp- tion of the former onto aluminum hydroxide and they made the additional observation that, freed of at least the greater part of the protein, the agent showed a considerably enhanced activity. They later reported the content of this concentrated extract to average 0.527 mg. per C.C.and reduc- ing substances 0.175 mg. per C.C. Guinea-pigs were not sensitized by injec- tions of this material. Lewis also modified her original conclusions when she found that the globulin was an unnecessary constituent of the filtrate (47). By adsorption of the protein with charcoal, tumor extracts were obtained yielding 0.83 mg. of nitrogen per 100 C.C.without obvious reduction in the tumor-producing properties of the partially purified material. Fraenkel (48) used numerous agents for adsorption and elution but was unable to effect a clean separation of agent and inhibitor, the former appearing in the super- natant fluid and as part of the adsorbed material. Pirie (49) conducted sim- ilar experiments and also showed that the partially purified agent, containing 10-hg. of nitrogen in the infective dose, is inactivated as rapidly and by means similar to those employed against the whole extract. A degree of purification has also been obtained by precipitation of the agent at pH 4 and resuspension at pH 8 by Sittenfield, Johnson and Jobling (50). Murphy and his coworkers precipitated the active material at a pH between 4.4 and 4.8 (51). Both groups interpreted their results to indicate that the agent had been separated from an inhibiting substance by this pro- cedure. Subsequent analytical studies on the purified agent by Murphy showed a nitrogen content of from 12.47 to 13.33 per cent, phosphorus of 0.22 to 0.27 per cent and reducing substances of 15.25 per cent figured as glucose. It having been learned that, although the agent can be precipitated with protein, it is equally active when freed of all detectable protein, it remained to investigate the other constituents of the filtrate for their tumor-producing activity. By the addition of gelatin at a pH 4.7 to the partially purified fil- trate, all demonstrable carbohydrate was removed by Claude, leaving the ac- tive principle in the supernatant fluid. Subsequent dialysis against running water rid the agent of further impurities and left an active material with a nitrogen content of 0.01 mg. per C.C. or about 7 per cent of the dry weight. Claude summarized the situation regarding purification in the following man- ner. " In contrast with this practically unreduced activity, usual chemical and biological tests for have become negative. An important con- taminating substance, a carbohydrate, can be removed without markedly re- ducing the activity. A certain number of remaining impurities can be elim- inated by further manipulations and, according to corresponding observations, the chicken tumor principle does not appear to give positive tests such as the ninhydrin test or the diazo reaction. Moreover, the agent is not affected by basic dyes such as neutral red, by barium chloride and is not precipitated by basic lead acetate " (52). To recapitulate, the chemical nature of some synthetic carcinogenic sub- stances, the close chemical relationship between estrogenic and carcinogenic materials and the chemical composition of the ovarian hormones, the effect of lipids upon growth of tissue, the high lipid content of tumors and other tissues of tumor-bearing subjects, and finally the fact that the agent of the Rous sar- coma can be largely separated from the protein and carbohydrate of the filtrate, all suggested the possibility of identification of the tumor-producing factor of the Rous sarcoma with the lipids. It has been our aim to apply the more direct methods of lipid extraction, testing the lipid fraction for its ability to reproduce the tumor.

The tumor used throughout these investigations, the Rous chicken sarcoma No. 1, was obtained from the Rockefeller Institute through the courtesy of Dr. Peyton Rous. Material for extraction is propagated by the routine in- oculation of fragments of tumor, suspended in normal saline, into both breasts of chickens. With rare exceptions the tumor grows progressively in all fowls, whether Rhode Island red, Plymouth Rock, or White Leghorn, and these 672 JAMES W. JOBLING, E. E. SPROUL AND SUE STEVENS three breeds have been used indiscriminately. As a rule the chicken receives the inoculation when from six to eight weeks of age and is killed by severing the neck two to two and a half weeks later. During this period the tumor has attained its maximum size, that from each breast weighing approximately 50 gm., and there is only a moderate degree of necrosis. If the chicken is allowed to die spontaneously at three to three and a half weeks, necrosis and hemorrhage are extensive. For all experiments, the entire tumor, both the degenerated and better preserved portions, was utilized, since these analyses were entirely qualitative and the necrotic tissue is capable of producing tumors. It seemed unlikely that we should be able to retain the activity of the agent in the lipid fraction if the technic of extraction were not adapted to the known limitations of the agent. An efficient extraction of total lipid calls for heat and requires a period of time prohibited by our knowledge of the in- stability of the tumor agent. It was demonstrated in early studies by Rous (53) that fifteen minutes at a temperature of 55" C. is sufficient for com- plete inactivation and that the potency of a filtrate cannot be maintained at 37" C. for longer than approximately twenty-four hours. All extractions and the process of removal of solvents were accordingly conducted either at room temperature or in a water bath regulated between 35 and 37" C. Where necessary the boiling point of the solvent was reduced to this level by use of a partial vacuum. In addition, the length of time from removal of the tumor to injection of the extract was reduced to a minimum, which, for the pre- liminary experiments, varied between about nine and twelve hours. It has also been shown that the activity of the filtrate is considerably en- hanced by lowering the oxygen tension of its environment or by the addition of certain reducing substances (54, 5 5, 56). Consequently the precaution was taken to replace the air in solution and container with nitrogen and at least a partial vacuum was maintained during the extraction and evaporation of the solvent. Preliminary Experiment: About 100 gm. of tumor tissue were removed without aseptic precautions and ground in an ordinary meat grinder. Further disintegration of the tissue was accomplished by vigorous grinding in a mortar with 100 C.C.of acetone. The mixture was then transferred to a flask and, despite the fact that the solvent restrains oxidation, nitrogen was bubbled through the suspension for fifteen minutes before shaking in a mechanical rotating device for one hour. The acetone was then decanted, separated from tumor particles by centrifugation and filtered through ordinary filter paper. Meanwhile the procedure was repeated with a similar quantity of acetone and the two extracts were combined. Evaporation of the solvent was effected in a vacuum distilling apparatus at approximately 35" C. until a gummy mass was obtained. A small stream of nitrogen was passed through the solution during evaporation. While this was in progress, the dried tumor was extracted for three hours with benzene at a temperature varying from 32 to 37" C. in a Soxhlet extrac- tion apparatus attached to the suction pump. The benzene extract was cen- trifuged, filtered clear, and added to the residue from the acetone extract in the distilling flask. Freed of benzene, the combined extract was dissolved in 30 C.C. of benzoinated lard, previously melted and cooled to 35' C., at which temperature it is still liquid. One cubic centimeter was inoculated immedi- ately into each breast of 13 chickens. The residue of the tumor, after acetone and benzene extraction, was ex- posed to a stream of nitrogen to remove the last traces of solvent, suspended in saline, and inoculated into 6 chickens. Eight chickens received 1 C.C.of benzoinated lard alone. Since it was our purpose, in these early experiments, to detect any activity, however reduced by the procedures employed, it was thought advisable to give repeated inoculations after the manner of those using the synthetic carcino- genic agents. Accordingly a fresh extract was prepared at approximately weekly intervals and injected at the same site. Results of Preliminary Experiments: Of the 13 chickens receiving the whole lipid extract, 6 died without gross evidence of tumor formation after 2 to 12 injections. Five died following 4 to 12 injections with large breast

TABLEI : Preliminary Experiment

Number Number Number Number Number with with Material Injected of 1 of Animals Injections Negative Malignant Benign Tumors Tumors

Acetone and benzene extract in benzoin- . ated lard 13 2-12 6

Residue after extraction 6 12 6

Benzoinated lard 8 12 8 tumors corresponding in size and appearance with those obtained by the usual methods of inoculation. Metastases were present in heart, lungs, and occa- sionally the liver and gonads. There .was no difficulty in transplanting 2 of these tumors (no attempt was made with the others) and one was carried through 27 transfers without reduction in activity. The 2 remaining animals died three months and a half after the first inoculation with circumscribed nodules from 1.5 to 2.5 cm. across, in each breast, but without metastases. Histologically all tumors were identical with the Rous chicken sarcoma. Sec- tions of the breast muscle from those regarded as negative frequently showed early growth of tumor tissue, but were still recorded as negative to avoid the criticism that granulation tissue might be confused histologically with a spindle-cell sarcoma. Neither the residue of dried partly defatted tumor nor the benzoinated lard alone produced tumors. The animals received a comparable number of injections. Further Purification of the Lipid Extract: Limited analyses were con- ducted on the extract obtained above, sufficient to indicate that a small quan- tity of protein was present, to which the activity might well be ascribed. Having discovered by another series of experiments that reextraction of the above material with petroleum ether yielded a product incapable of producing 674 JAMES W. JOBLING, E. E. SPROUL AND SUE STEVENS tumors, it was decided to inject a sample following each step in the preliminary procedure in order to discover which extract carried the active material. The plan of extraction was essentially similar with the following altera- tions. A larger quantity of fresh tumor tissue was used, between 300 and 400 gm., depending on the amount immediately available, and the quantity of solvent was correspondingly increased. In one experiment the entire ace- tone fraction obtained by combination of the first and second acetone extracts was divided into two equal parts. Half was concentrated with the usual precautions and the watery residue, about 30 c.c., was inoculated into the breasts of 10 chickens. The second half of the acetone fraction was also freed of acetone and the residue reextracted by shaking in a flask with three changes of benzene for half an hour each. Nitrogen was again forced through the mixtures at each step. The benzene extract of the acetone fraction was combined with the benzene extract of the tumor obtained as before in the Soxhlet, filtered clear, evaporated under negative pressure to dryness, and in- oculated into the breast muscle of 12 chickens. Benzoinated lard was not used in this experiment since previous experience showed that it formed a solid mass, slowly absorbed, and it was felt that this might prevent direct contact of the agent with the muscle. Consequently about 20 C.C.of distilled water were added to the flask to form an emulsion of the benzene extract. The plan of repeated inoculations at weekly intervals was again followed. The results are shown in Table 11. In the first group, tumors began to appear after 6 injections. Eight of the 10 chickens died within two months with large breast tumors and extensive metastases in lungs and heart. One died at the end of five weeks with no tumor. The remaining chicken con- tinued to live and was killed eight months later. Those chickens receiving the benzene extract fared poorly. Six died after from 2 to 12 injections in an extreme state of malnutrition and without evidence of tumor growth. Their poor condition was attributed in part to the difficulty in removing the last traces of benzene from the extract. The remaining 6 were allowed to live without further inoculations. One .died after four months, one after six months, without tumor. To test their susceptibility, the last 4 chickens were injected with tumor tissues which grew steadily and killed all after the usual course. In another experiment conducted during the same period, the first and second acetone extracts were not combined, but were injected individually after removal of the solvent to locate more accurately the active principle. Half of the first acetone fraction was further divided by extraction with three changes of benzene under nitrogen, as before, and both the benzene extract and the residue therefrom were inoculated separately. The results of this and the preceding experiment are recorded in Table 11. Among those chickens receiving the first acetone extract, one died after three injections with atrophic muscle and no tumor. Eleven of the 13 fowls died within two months, having received 5 inoculations, disclosing large breast tumors, histologically identical with the Rous sarcoma No. 1 and accompanied by extensive metastatic involvement of lungs and heart. The chickens in- jected with the second acetone extract, the benzene extract and the residue from the latter, were negative. These animals were tested after an interval of five months to see if they were susceptible to the tumors. No significant resistance was demonstrated.

TABLEI1 : Sepnration of Frartions -- - - Number Number Material Injected Number of Number of Animals Injections Negative Positive -- Combined acetone extract 10 5-8 2 8

Benzene extract of above and of tumor 12 2-16 12 0

First acetone extract 13 3-5 2 11

Second acetone extract 14 8 14 0

Benzene extract of first acetone extract 15 8 15 0

Residue from above 14 8 14 0

Substitution of Carbon Tetrachloride for Benzene: At this point in the investigation the only positive finding was the presence of the agent in activc form in the first acetone extract which was known to have small quantities of substances other than lipid, presumably carried along in the water removed from the tumor. It remained then to demonstrate activity in an extract en- tirely free of protein and carbohydrate, but all attempts to do so had failed. There seemed to be three possible explanations for this failure; first, that the agent was not a lipid; second, that the method was too drastic and that an active lipid fraction might be obtained by improvements in technic; and lastly, that while the lipid might be the specific cancer agent it was too unstable when dissociated, say, from some lipo-protein . Before proceeding with the third hypothesis it seemed advisable to try a solvent other than benzene. Carbon tetrachloride was selected as one ob- tainable in a pure state and having little oxidizing proclivity. The procedure of extraction was not varied in any other detail; again great care was taken to avoid exposure to air, the temperature was regulated below 37" C., and the entire experiment required approximately ten hours. Results: All 4 chickens receiving .the watery residue of the combined acetone extracts developed tumors after two injections. The tumors were large and metastases numerous. The second group of 10 animals inoculated with the carbon tetrachloride fraction failed to develop tumors. Three died with atrophic muscle and infection about the eyes and in the throat. The remainder received 12 injections at weekly intervals without effect. We see then that our experience with the benzene extract was repeated when carbon tetrachloride was used as an alternative solvent. Combination of the Tumor Lipid with Non-Tumor Tissue Extract: At- tention was then directed toward the hypothesis that the lipid required the presence of some other substance, possibly a protein, which was not neces- sarily derived from the tumor. The lipid was prepared as before by two ex- TABLEIV: Acetone and Carbon Tetrachloride Extraction (Results from 11 experiments)

\Vet Volume rota1 Solids Solids Total Total Reducing Tissue Per Cent Extracted Extracted Nitrogen Phosphorus NIP* Substance Extracted Acetone (per cent (dry wt .: (per cent solids (per cent solids (per cent solids in Extract wet tissue) mg./100 c.c.) extracted) extracted) extracted) -. (grams) -- First acetone extract High 522 57.4 Low 272 45.5 Average 349 49.9

Second acetone extract High Low Average

Carbon tetrachloride ex- tract High Low .4verage

* The high and low NIP given are those found in the series of experiments and do not correspond to the high and low N and P given in their respective columns. t See djxussion under Results tractions of the fresh tumor with acetone, removal of the solvent, and re- extraction of this as well as of the dry, partly defatted tumor with benzene or, at a later date, with carbon tetrachloride. The final benzene or carbon tetrachloride extract was filtered clear. For an extract of non-tumor tissue it was thought advisable to obtain the tissue most closely related to the sarcoma. From the histological appearance of the tumor one would surmise that the cells were derived from connective tissue. Duran-Reynals and Murphy (57) showed that connective tissue could " fix " the agent, a property which no other tissue possessed. Carrel (58) believed that spindle cells could be derived from monocytes and that the Rous tumor agent was instrumental in bringing this about. With these ob- servations in mind the extract of non-tumor tissue was prepared in the follow- ing manner. A normal chicken was inoculated on three successive days with a saline suspension of kieselguhr, a diatomaceous earth often used to cause tissue in- jury and known to be incapable of producing tumors. The kieselguhr was

TABLE111 : Combination of Benzene Extract of Tumor with Saline Extract of Non-tumor Tissue

Material Injected Number of Number of Number Number Injections Animals Negative Positive

Combined acetone extract 3-4 4 1 3 Benzene extract tumor and saline extract kiesel- guhr exudate 8-1 1 12 6 6 injected in all portions of both breasts. In order to have exudate of recent formation as well as young scar tissue, the last inoculation was performed on the morning the experiment was conducted. Section through muscle thus treated showed soft yellow and yellowish gray moist zones which could be dis- sected from the better preserved muscle and utilized for this experiment. Microscopically such tissue is composed of foreign-body giant cells enclosing particles of earth. There are areas of more spindle-shaped cells and many mononuclear wandering cells are present. This tissue was ground in a mortar with washed sand and a sufficient quan- tity of normal saline to make a 5 per cent suspension. The heavy particles were removed by centrifugation and the supernatant fluid was used for the normal tissue extract. The benzene extract of tumor which was being pre- pared at the same time was suspended in a quantity of saline equal to that used in preparation of the non-tumor extract. The two were combined and nitrogen bubbled through vigorously at 37' C. for one hour. The mixture was roughly divided to provide a dose for each breast of 12 chickens. Weekly injections of freshly prepared material were given. To be certain that the positive results previously obtained could still be repeated, 4 chickens received a portion of the acetone extract from the same tumors providing the benzene fraction. Results: One chicken receiving the acetone extract died with infection about the eyes. The remaining three developed very large tumors filling TABLEV: Acetone and Benzene Extractions (Results from 14 experiments)

Wet Volume Total Solids Solids Total Total Reducing Tissue Per Cent Extracted Extracted Nitrogen Phosphorus Substance Extracted Acetone (per cent (dry wt.: (per cent solids (per cent solids (per cent solids (grams) in Extract wet tissue) mg./100 c.c.) extracted) extracted) extracted)

First acetone extract High Low s Average od Second acetone extract High Low Average

Benzene extract High Low Average I *See footnote to Table IV. t See discussion under Results. the breasts and lungs. The group receiving the benzene extract of the tumor mixed with the saline extract of non-tumor tissue were also positive. Of the 12 injected, 6 died before the appearance of any tumor, showing infection or emaciation, occasionally with necrosis of the liver. The remaining 6, that is, all those living over four weeks from the beginning of the experiment, were found to have tumors comparable to the ones ordinarily produced by routine inoculation. Metastases were conspicuous. The tumors first appeared in this group two months after onset of the experiment, and grew rapidly, causing death of the animal in two to two and a half weeks. The above experiment was repeated using carbon tetrachloride in place of benzene with comparable results.

Analytical studies were conducted by one of us (S. S.) on portions of the same lipid extracts which were injected into the chickens with the above re- sults. These chemical analyses served the double purpose of determining the uniformity of the extracts and providing information concerning those substances associated with the active principle in such extracts. Since com- plete extraction of the lipids was never possible by the method described, the following data do not represent an analysis of the entire lipid content of the sarcomata. Chemical Methods Acetone Determination: The amount of acetone present in the first and second acetone extracts was determined by Hubbard's modification of Shaf- fer's iodometric procedure (59). The water removed from the tissue may be calculated by subtraction from the total volume. Total Solids Extracted: This refers to the total amount of material ex- tracted by a given solvent. An aliquot was measured into a previously weighed weighing bottle, placed in another bottle in a water bath at 40-60' C. and evaporated with the aid of suction and nitrogen. The residue remained in an evacuated desiccator with calcium chloride over night and was weighed. These operations were repeated until constant weight was obtained. Nitrogen Determinations: The total nitrogen present was found by a modified micro-Kjeldahl procedure. The distilling apparatus used was Goebel's (60) modification of Pregl's. In place of the usual HC1 or H,SO, to retain the distilled , a saturated solution of boric acid, with methyl red as an indicator, was used (61). Nitrogen was determined on the material extracted by acetone and coag- u.lated by 16 per cent trichloracetic acid. After filtration, the nitrogen was determined on the precipitate by the procedure described above. Total Phosphorus Determinations: After acid digestion of an aliquot, the Fiske and Subbarow (62) colorimetric procedure was followed. Total Reducing Substances: These include those substances which, after hydrolysis with hydrochloric acid, will reduce alkaline potassium ferricyanide to ferrocyanide. The colorimetric micro-ferricyanide method of Folin (63) was used and the results were calculated as glucose. m N m3 I- h WOI 'Qv) '0-4' 0.3 'QQ wv) 3 Results Tables IV and V summarize the analyses of the extracts which were in- oculated into chickens in the preliminary experiments. There is considerable variation, due in part to the lack of uniformity of the tumors and in part to the incomplete extractions. The total solids extracted by carbon tetrachloride and benzene were extremely variable. This was largely attributable to the fact that the second acetone extract was soluble in these solvents and incon- stant amounts of the former were removed for inoculation and analysis. The findings listed under this heading are submitted merely to show the amount of material which was obtained for inoculation and analysis. The first acetone extract contained a small amount of material coagulable by 16 per cent trichloracetic acid. The nitrogen value of this precipitate showed for the acetone-carbon tetrachloride series an average of 0.28 per cent (high 0.37 per cent and low 0.09 per cent). This lxtract, it will be re- called, was the only one showing activity without addition of a normal muscle extract. The presence of a small amount of protein may account for this fact.' Two experiments were carried out later for the purpose of determining the amount of cholesterol and phospholipid in the three different extracts, carbon tetrachloride being used as the last solvent. The two acetone extracts were evaporated to dryness with the aid of negative pressure and nitrogen and then extracted with a 3:l alcohol-ether mixture. Aliquots of the latter were used for cholesterol and phospholipid determinations. The Schoen- heimer and Sperry (64) micro-method, modified for colorimeter, was applied for cholesterol estimation and Bloor's (65) oxidative procedure for phos- pholipid. The results are given in Table VI. In determining the amount of cholesterol in the first acetone extract an aliquot of the alcohol-ether extract equivalent to 40 C.C.of the former was used. The final result was a slight green color, which has been called a " trace " in Table VI. For the phospholipid content of the first acetone ex- tract, an alcohol-ether aliquot equivalent to 20 C.C. of the original extract was used. The former, if present, was too slight to be estimated by this method.

The experiments reported in this paper represent our earliest attempts to isolate the agent responsible for the transmission of the Rous chicken sar- coma No. 1, following the assumption that it was related chemically to the lipids. They are not offered as methods worthy of repetition, subsequent work having developed much more effective means of extracting an active lipid fraction from the tumor, but they represent procedures likely to be used by anyone with a similar purpose and as such should be of value for comparative purposes. Tumors similar in all details to the original tumor were obtained by in- jection of an acetone extract which was shown to contain a small quantity of protein as well as the lipid. A more purified extract obtained by use of benzene or carbon tetrachloride was incapable of transmitting the tumor when injected alone, but if incubated for a brief time with a saline extract of the normal tissue from which the tumor is thought to arise, it reproduced the 682 JAMES W. JOBLING, E. E. SPROUL AND SUE STEVENS tumor. These more purified extracts were shown to have a low nitrogen content, averaging 0.55 per cent of the solids extracted when benzene was used and 0.56 per cent with carbon tetrachloride. Reducing substances were correspondingly low. In all the foregoing experiments it seemed requisite to repeat the injec- tions. In view of subsequent success in producing tumors with one inocula- tion it is probable that such measures were superfluous and that a single in- jection actually initiated the growth of the tumors.

NOTE:We are deeply grateful to Dr. Hans Clarke and Dr. Rudolph Schoenheimer for their kindly advice and interest during this investigation.

1. Rous, P.: A sarcoma of the fowl transmissible by an agent separable from the tumor cells, J. Exper. Med. 13: 397, 1911. 2. ANDREWES,C. H.: Viruses in relation to the etiology of tumors, Lancet 2: 63 and 117, 1934. 3. Rous, P.: The virus tumors and the tumor problem, Am. J. Cancer 28: 233, 1936. 4. LEDINGHAM,J. C. G., AND GYE,W. E.: On the nature of the filterable tumor-exciting agent in avian sarcomata, Lancet 1: 376, 1935. 5. WELLS,H. G.: The nature and etiology of cancer, Am. J. Cancer 15: 1919, 1931. 6. KNOX,L. C.: Trauma and tumors, Arch. Path. 7: 274, 1929. 7. EWING,J.: The modern attitude toward traumatic cancer, Arch. Path. 19: 690, 1935. 8. HARDING,W. G.: Trauma and tumor pathogenesis, J. Cancer Res. Com., Univ. Sydney 7: 81. 1935. 9. BRISTOL,L. D.: An enzyme theory of cancer etiology, M. Rec. 89: 180, 1916. 10. ENGEL,P.: Wachstum~beeinflu~~endeHormone und Tumorwachstum, Ztschr. f. Krebs- forsch. 41: 488, 1935. 11. JAISOHN,P.: DO normal cells change to cancer cells under hormonal influence? West Virginia M. J. 30: 414, 1934. 12. BOURNE,G.: Sterols, sex hormones and cancer, J. Cancer Res. Com., Univ. Sydney 7: 34, 1935. 13. MURPHY,J. B.: The experimental approach to the cancer problem; Bull. Johns Hop- kins Hosp. 56: 1, 1935. 14. LUDFORD,R. J.: The somatic cell mutation theory of cancer, ~inthSc. Report, Im- perial Cancer Research Fund, 1930, p. 121. 15. GYE,W. E., AND PURDY,W. J. : The Cause of Cancer, Cassell & Co., London, 1931. 16. COOK,J. W., HEWETT,C. L., AND HIEGER,I.: The isolation of a cancer producing hydro- carbon from coal tar, J. Chem. Soc. No. 106: 395, 1933. 17. COOK,J. W.: The production of cancer by pure hydrocarbons, Proc. Roy. Soc., London, ser. B, 111 : 485, 1932. BARRY,G., COOK,J. W., ET AL:The production of cancer by pure hydrocarbons, Proc. Roy. Soc., London, ser. B, 117: 3 18, 1935. 18. COOK,J. W., ET AL: Chemical compounds as carcinogenic agents, Am. J. Cancer 29: 219, 1937. 19. FIESER, L. F.: The of Natural Products Related to Phenanthrene, Reinhold Publishing Corp., New York, 1936, Chapter 111, p. 81. 20. LOEB,L.: Estrogenic hormones and carcinogenesis, J. A. M. A. 104: 1597, 1935. 21. TAYLOR,H. C., JR.: The evidence for an endocrine factor in the etiology of mammary tumors, Am. J. Cancer 27: 525, 1936. 22. LATHROP,A. E. C., AND LOEB,L.: Further investigations on the origin of tumors in mice. Part 111. On the part played by internal secretion in the spontaneous de- velopment of tumors, J. Cancer Research 1 : 1, 1916. 23. COOK,J. W., AND DODDS,E. C.: Sex horinones and cancer producing compounds, Na- ture 131: 205, 1933. 24. LACASSAGNE,A.: Apparition de cancers de la mamelle chez la souris mkle soumise i des injections de iolliculine, Compt. rend. Acad. de sc. 195: 630, 1932. 25. COOK,J. W., DODDS,E. C., HEWETT,C. L., AND LAWSON,W.: The estrogenic activity of some condensed ring compounds in relation to their other biological activities, Proc. Roy. Soc., ser. B, 114: 272, 1934. 26. ROBERTSON,T. B., AND BURNETT,T. C.: The influence of lecithin and cholesterin upon the growth of tumors, J. Exper. Med. 17: 344, 1913. 27. BERNSTEIN,S., AND ELIAS,H.: Carcinomwachstum und Lipoide, Med. Klin. 23: 1616, 1927. 28. MORAVEK,V.: The influence of cholesterol and lecithin on tumors. 11. Rous chicken sarcoma, Arch. Clin. Cancer Research 4: 129, 1929. 29. SWEET,J. E., CORSON-WHITE,E. P., AND SAXON,G. J.: Further studies on the relation of diet to transmissible tumors, J. Biol. Chem. 21: 309, 1915. 30. HARNES,A. R.: The influence of certain lipoids on the growth of a rabbit neoplasm, J. Lab. & Clin. Med. 20: 1077, 1935. 31. VASSILIADIS,H.: AU sujet des substances organiques activant la pousse des cancers experimentaux, Compt. rend. Soc. de biol. 118: 1483, 1935. 32. MACFADYEN,D. A., AND STURM,E.: Further observations on factors from normal tis- sues influencing the growth of transplanted cancer, Science 84: 67, 1936. 33. BULLOCK,W. E., AND CRAMER,W.: Contributions to the of growth; on the lipoids of transplantable tumors of the mouse and rat, Proc. Roy. Soc. London, ser. B, 87: 236, 1914. 34. BIERICH,R., AND LANG,A.: Uber den Lipoidgehalt bijsartiger Geschwiilste, Ztschr. f. physiol. Chem. 216: 217, 1933. 35. YASUDA,M., AND BLOOR,W. R.: The lipid content of tumors, J. Clin. Investigation 11: 677, 1932. 36. HAVEN,F. L.: Tumor lipids: the comparative lipid content of the periphery and center, Am. J. Cancer 29: 57, 1937. 37. ROFFO,A. H., AND THOMAS,J.: La chimie du cancer, Paris, Vigot Frkres, 1933. 38. CORREA,L. M.: LOSlipidos en el cbncer, Boll. Inst, de med. exper. para el estud. y trat. d. cbncer 10: 511, 1933. 39. BURGHEIM,F., AND JOEL,W.: Uber Beziehungen zwischen Krebs und Lipoidstoffwechsel, Klin. Wchnschr. 10: 397, 1931. 40. LE MAY,P.: Lipoides et cancer, NCoplasmes, 10: 158, 1931. 41. JOWETT,M.: The phosphatide and cholesterol content of normal and malignant human tissues, Biochem. J. 25: 1991, 1931. 42. TESAURO,G.: Zur Frage der Beeinflussung des Tumorwachstums durch Lipoide, Ztschr. f. Krebsforsch. 35: 269, 1932. 43. BRIKKER,F., AND LASARIS,J.: Zur Lipoidcharakteristik des Krcbsgewebes, Ztschr. f. Krebsforsch. 37: 432, 1932. 44. LEITCH,A.: 011 the pathogenesis of cancer, Report of International Conference on Cancer, London, 1928, p. 20. 45. LEWIS,M. R., AND MENDELSOHN,W.: Salting out the causative agent of chicken tumor with the globulin fraction of the Berkefeld filtrate of the tumor, Am. J. Hygiene 12: 686, 1930. 46. MURPHY,J. B., HELMER,0. M., CLAUDE,A., AND STURM,E.: Observations concerning the causative agent of a chicken tumor, Science 73: 266, 1931. 47. LEWIS,M. R., AND MENDELSOHN,W.: Purified (protein free) virus of chicken tumor No. 1, Am. J. Hygiene 13: 639, 1931. 48. FRAENKEL,E. M., AND MAWSON,C. A.: Adsorption and elution of the Rous agent, Brit. J. Exper. Path. 16: 416, 1935. 49. PIRIE, A.: Adsorption experiments with the Rous sarcoma virus, Brit. J. Exper. Path. 12: 373, 1931. 50. SITTENFIELD,M. J., JOHNSON,B., AND JOBLING,J. W.: The demonstration of inhibitory substances in the filtrate of the Rous chicken sarcoma and their separation from the active agent, Am. J. Cancer, 15: 2275, 1931. 684 JAMES W. JOBLING, E. E. SPROUL AND SUE STEVENS

51. MURPHY,J. B., STURM,E., CLAUDE,A., AND HELMER,0. M.: Properties of the causa. tive agent of a chicken tumor. Attempts at isolation of the active principle, J Exper. Med. 56: 91, 1932. 52. CLAUDE,A,, AND MURPHY,J. B.: Transmissible tumors of the fowl, Physiol. Rev. 13: 246, 1933. 53. Rous, P., AND MURPHY,J. B.: Nature of the filterable agent causing the sarcoma of fowl, J. A. M. A. 58: 1938, 1912. 54. MUELLER,J. H.: The effect of oxidation of filtrates of a chicken sarcoma (chicken tumor I-Rous), J. Exper. Med. 48: 343, 1928. 55. PIRIE, A., AND HOLMES,B. F.: The cause of inactivation of the Rous chicken sarcoma during incubation, Brit. J. Exper. Path. 12 : 127, 1931. 56. GYE,W. E., AND PURDY,W. J.: The Rous sarcoma No. 1. Loss of filtrate activity at incubator temperature: Protection by means of hydrocyanic acid, Brit. J. Exper. Path. 11: 282, 1930. 57. DURAN-REYNALS,F., AND MURPHY,J. B.: Properties of the causative agent of a chicken tumor. The specific fixation by tissues of susceptible animals, J. Exper. Med. 50: 315, 1929. 58. CARREL,A., AND EBELING,A. H.: The transformation of monocytes into fibroblasts through the action of the Rous virus, J. Exper. Med. 43: 461, 1926. 59. PETERS,J. P., AND VAN SLYKE,D. D.: Quantitative Clinical Chemistry, Williams & Wilkins Co., Baltimore, 1932, Vol. 11, p. 636. 60. Ibid: p. 530. 61. MEEKER,E. W., AND WAGNER,E. C.: Ind. and Eng. Chem. Anal., Ed. 5: 396, 1933. 62. FISEE, C. H., AND SUBBAROW,Y.: The colorimetric determination of phosphorus, J. Biol. Chem. 66: 375, 1925. 63. FOLIN, OTTO: Laboratory Manual of Biological Chemistry, 5th Ed., D. Appleton- Century Co., New York, Ed. 5, 1934, p. 307. 64. SCHOENHEIMER,R., AND SPERRY,W. M.: A micromethod for the determination of free and combined cholesterol, J. Biol. Chem. 106: 715, 1934. 65. BLOOR,W. R.: The oxidative determination of phospholipid (lecithin and cephalin) in blood and tissues, J. Biol. Chem. 82: 273, 1929.