The relatively short survival time of animals bearing transplanted tumors has been a handicap for the study of the influence exerted by many agents. Effects requiring some time before they can be induced are thus missed. Changes which occur in tumors—such as the tendency to ulcerate after treatment with fatty acids—have been found to be transmitted in successive generations of the tumors. This has led us to carry on treatment beyond the survival time of one individual host in order to study the influence of various agents. In one group of experiments, this was done through treatment of the successive hosts of serial transplants. In another group of experiments, the treatment was applied to the transplants themselves in successive hosts.

Mice with grafted tumors were treated with the chosen agents. When the tumor in a treated host, or in a control, had grown to 1 1/2 centimeter diameter, it was removed. Part of it was used for further transplants, part for microscopic studies. The rest of the animals were kept until death and the survival time was noted. Transplants of the tumor from treated animals as well as from controls were grafted in new animals and the treatment continued for the new hosts. This procedure was repeated for successive generations. In other experiments, the successive transplants were dipped, prior to grafting, in an oily solution or in a suspension in saline of the agent being tested. The procedure was repeated continuously for both treated animals and controls, and growth and survival time were noted. The following experiments are characteristic.

Using the insaponifiable fractions of human placenta in an oily solution of 5%, or in a saline suspension corresponding to two milligrams of the material per cubic centimeter, the following results were observed in the case of Ehrlich mammary carcinoma in mice. No changes in survival time, evolution of the tumor, gross or microscopic character were seen in the first and in some experiments even in the second generation. Usually with the third generation, the survival time was reduced, the tumor growing much more rapidly and killing the animal in around 20 days. The malignant character of the tumor was seen to increase in the subsequent transplants and in the fifth generation in some experiments, killed the animal in less than a week. The morphological change observed in these successive transplants were also characteristic. The tumor was seen to change from solid to encephaloid. The adenocarcinomatous character was thus altered and the degree of undifferentiation was increased by passing through the third, fourth and sixth generation. At the sixth generation in some experiments—and the fifth or eighth in others—microscopic examination showed that sarcomatoid portions were present in the tumor. The malignancy appeared to be at its maximum in these tumors. Transplants of tumors with sarcomatoid microscopic character, if treated in the same manner, gave negative grafts. Thus, it appears that the treatment with the insaponifiable fractions has progressively increased the malignancy until the moment when sarcomatous character appeared after which negative transplants were observed.

Spectral analysis of the insaponifiable fraction of various origins

Fig. 294. Spectral analysis of the insaponifiable fraction of various origins. It shows a characteristic peak at 450 mu, another at 360 and another at 272. The organs in which they are especially present are indicated.

The treatment of a tumor with lipoacid preparations of human placenta has produced opposite changes manifest even in the first transplants. These increased in the second generation. After the second and very rarely after the third grafts, negative transplants were obtained. We used this method of treating tumors through successive generations routinely. The results obtained for different agents are discussed in the text of this publication.

Details of the spectral analysis of rat

Fig. 295. Details of the spectral analysis of rat colostrum indicates the existence of a formation with three peaks in the region 290-250 mµ with some similarity to the conjugated trienes.

Chapter 13, Note 7. Conjugated Trienic Alcohols

Spectral analysis has permitted us to recognize the presence, in certain mixtures of the insaponifiable fraction, of several peaks, some especially interesting. Characteristic peaks were seen at 450, 360 and 272 mµ, as shown in Figure 294. In the first analysis, one was identified as corresponding to a peak of 2720 Angstroms. In more complete further spectral analyses, it could be seen to correspond to a conjugated triene with its characteristic three peaks. The fact that it corresponds to a substance with positive polar group explains why, compared with conjugated acids, the curve shows a marked displacement of the peaks toward higher wave lengths. (Fig. 295) This can be related to the different influence exerted by the electrically opposite polar groups. This compound was first found in the colostrum obtained from the stomach of newborn rats on the first day. In smaller amounts, it has been seen in other samples of milk or butter, and in pork kidneys. It has been found less frequently in growing tumors and is even rarer in growing animals.

The same spectral analysis has permitted us to recognize other peaks and relate them to the different sources from which the unsaponifiable fraction was obtained. Fig. 294 shows these peaks and indicates their correlation with the origin of the material.