HOLLAND N. STEVENSON From Columbia University, George Crocker Special Research Fund, F. C. Wood, Director. Received for publication June 7, 1917

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1 , TUMOR IMMUNITY IN THE CHICK EMBRYO HOLLAND N. STEVENSON From Columbia University, George Crocker Special Research Fund, F. C. Wood, Director Received for publication June 7, 1917 In a recent communication, experiments were reported in which it was found that adult chicken spleen had no influence upon the growth of mouse and rat tumors in the chick embryo, The tumor and spleen were inoculated simultaneously and allowed to grow for ten days, and the volume of spleen introduced (0.005 gram) exceeded the quantity of tumor (0.003 gram). The amount of spleen was determined arbitrarily, by the dose of mouse spleen necessary to induce immunity in the mouse and also by the comparative size of the seven-day chick embryo and the adult mouse; it appeared that the quantity of spleen used would be more than enough to induce immunity in the chick embryo if this were possible. It was found, however, that although over one thousand fertile eggs were inoculated, with six tumors of varying characteristics, the grafts proliferated without any suggestion of inhibition. The presence of the splenic graft on the allantois induced a striking increase in the round-cell reaction about the tumors; on careful study, however, it was found that the majority of these cells were of the myeloid or granular leucocyte series, and not lymphocytes. It was pointed out in the paper, in discussing these findings, that, although this reaction had been induced and yet had not influenced the growth of the tumor, it would be necessary to determine whether or not even larger doses of spleen would prevent the tumor from growing. The observations on the proliferative power of tumors growing in the presence of adult chicken spleen had been based upon a study of the grafts in serial 449

2 ~ spleen 450 HOLLAND N. STEVEN SON section, tumors in the sections containing spleen being adjudged as living when mitotic figures were found. But in the discussion of an abstract of the paper just referred to, which was presented before the American Association for Cancer Research at its annual meeting in April, 1917, GaylQrd pointed out that a tumor showing mitotic figures is not always alive, and that for this reason one would be unable to say, with absolute certainty, whether these tumors growing in the presence of spleen were or were not viable. Although the presence of numerous mitotic figures in a uniformly healthy tumor showing no necrosis is the usually accepted criterion of its viability and malignancy, Gaylord s criticism is not entirely unjustified in this instance; for while the slides presented at the meeting showed that the tumors fulfilled all the requirements necessary to establish their malignancy microscopically, it was impossible at the time to present further evidence in corroboration. The following experiments were therefore carried out, to determine : 1. Whether or not adult chicken spleen in amounts greater than gram will influence the growth of a tumor when both are inoculated simultaneously into the chick embryo. 2. Whether a rat tumor, after growing in the presence of in the chick embryo for ten daye, and showing mitoses, is alive, as demonstrated by its capacity for continued proliferation in the rat. EXPERIMENTS The conditions of experimentation were similar to those previously reported; the eggs were derived from the same source, and the technique of inoculation was similar to that employed previously. Inoculations were made on the seventh day of incubation and the grafts removed on the seventeenth or eighteenth day of incubation. 1. The spleen was removed from healthy adult fowls, minced with scissors, drawn up into a finely graduated syringe, and inoculated in amounts of 0.02 cc. upon the allantois of seventy-

3 TUMOR IMMUNITY IN THE CHICK EMBRYO 451 nine embryos, together with gram of a healthy Jensen rat sarcoma, the same tumor used by Dr. J. B. Murphy in his experiments. Twenty-two control eggs were injected with a similar amount of tumor alone. Ten days later the eggs were opened, and the grafts removed; of the controls, fourteen were living, and of these eight bore tumor grafts. The tumor, in serial sections where both tumor and spleen were present, was in a healthy growing condition and contained many mitotic figures. The spleen grafts (fig. l), as a rule, showed large healthy areas, but there were also large necrotic,., FIG. 1. SPLENIC GRAFT ON THE -4LLANTOIS Arising from the inoculation of 0.02 cc. of splenic mush. Jensen rat sarcoma is seen at one end of the graft. X 10. A small portion of areas present, as is generally the case when very large quantities of tissue of any type is inoculated. No difference in the reaction on the part of the wandering cells (fig. 2), which were abundant, or in the condition of the tumor, was noted on comparison with those obtained previously with the smaller dosage of spleen. On comparing these grafts with the controls, no difference in their condition could be discovered. 2. A series similar to the above was inoculated, using gram of a Jensen rat sarcoma and 0.02 cc. of a preparation of adult chicken spleen in each egg. A control series of eggs was inoculated with the tumor alone. When the grafts were re-

4 452 HOLLAND N. STEVENBOM. moved ten days later, seven of the largest of those growing with spleen were taken out aseptically from the egg, and each was cut into two parts, one of which was inoculated into twelve rats, the other being saved for microscopic examination. One of the grafts resulting from the control series of eggs was treated in a similar way. The boxes and the portion of the graft saved for section were given identical numbers, FIQ. 2. JINSEN EAT SARCOMA FROM 1 Shdwing the wandering cell reaction and numerous mitotic figures in the tumor cells. X 300. Tumors (fig. 3) developed in three of the series of rats and in the control; these were removed, their identity proved by sections, and one of them was inoculated into a second series of rats, where normal growth resulted. The chart (fig. 4) shows the growth of the tumors in the rat, after growing with spleen and without spleen in the chick; there is no difference between the two series.

5 TUMOR IMMUNITY IN THE CHICK EMBRYO 453 FIG. 3. JENSEN RAT SARCOMA IN THE RAT Resulting from the inoculation of a portion of the graft shown in 1 and 2. x 300. TUMORS IN RAT

6 454 HOLLAND N. STEVENSON Examination of sections of those grafts which had grown in the chick embryo in the presence of spleen, and from which the inoculations were made, was of interest, Some of these contained merely the tumor in a healthy condition, while 0 t h ~ ~ contained both tumor and spleen. The grafts from which tthe three series of rats that bore tumors were inoculated, showcrl in the sections large healthy grafts of spleen. Another experiment similar to this was carried out, using adult chicken spleen in the amount of 0.01 cc. and the Jensen rat sarcoma. In this series, also, the inoculations into rats of grafts that had grown with spleen in the chick, developed a certain number of tumors. The sections of the grafts, frorri which these tumors resulted in the animal, contained splenic grafts in a healthy viable condition. SUMMARY There appears to be little necessity for a discussion of the results of the experiments presented above, The total number of fertile eggs employed was about three hundred. The results were uniform, and prove that the tumors exhibited no signs of inhibition when they were grown on the allantois of the chick embryo for ten days, in the presence of adult chicken spleen in amounts up to 0.02 cc. This was demonstrated not only by the microscopical appearance of the tumors, but by their unmodified growth when returned to the animal to which they are native.