It has been indicated that the milk tumor agent in culture medium without cells at 37° C. disappears within an hour. The fact, however, that the virus persists for months in cell culture with repeated fluid changes and still produces so many tumors certainly suggests that something has happened. But this may not necessarily mean that the stability of the virus in the culture containing cells is the same as that in the medium without cells.

I am led to this comment because several years ago when we tried to grow certain strains of polio virus in chick-embryo tissue cultures, we quickly found ourselves in a similar situation. The virus incubated in the medium without cells disappeared shortly, but the agent persisted for several weeks longer in the tubes containing the chick-embryo tissue. Furthermore, we would wash the cells and think that we had gotten rid of everything by dilution. After the cultures were in the roller drum for a time, there was the virus again. An explanation was related simply to a change in the pH produced by the metabolizing cells. When the pH was properly adjusted, the polio virus persisted at 37° C. without chick-embryo cells as well and as long as in cultures of the cells.

Thus it was learned that repeated washings did not remove virus from the cells as calculated by directly simple dilution, but that a certain amount remained adsorbed either through the plasma clot or on the cells, and it kept coming off.

Then it has been shown recently that certain amino acids increase the thermal stability of poliomyelitis virus. Thus it would seem necessary to control studies with respect to the possibility that either the pH produced by the metabolizing cells or certain amino acids liberated into the medium by the metabolizing cells (and perhaps they are even liberated by a stroma and not as much by epithelial cells) may in themselves contribute a more appropriate medium for stability of the virus. This might be tested by measuring the stability of the virus in the fluid from the control cultures, without the tumor agent, to see if there is a difference in the stability of the agent diluted in culture fluid from that of the agent diluted with fluid in which no cells had been grown. This should provide an informative control.

I was particularly impressed by the data that Dr. Alice Moore discussed. This business of a little virus coming along is very disheartening-now you see it; now you don't-and it goes on for 2 months, then 2 years. The stick-to-itiveness of these people calls for much admiration. These results definitely suggest virus synthesis at a level which is barely detectable. Now, again, I mention that the kind of pattern Dr. Moore described (and it may also apply to the data of Dr. Lasfargues) has been found with so-called classic viruses when either the pH of the medium or the temperature (there may be other factors) is just wrong. If a virus that multiplies optimally at 37° C. is cultured at a temperature only 2° higher, one may observe just this pattern. Or, with the reverse, if the temperature is decreased a few degrees, such a pattern may result. Now, it may be that the temperature of the mammary tissue is not like the 38° C. of the normal mouse. Perhaps it would be desirable to move the temperature down to 34° or to 30° C, and maybe, at those temperatures, more virus would be formed.

Dr. Sanford (National Institutes of Health): With Dr. Andervont (National Institutes of Health) we have been trying to learn whether the mammary-tumor agent can be maintained in long-term cultures of the mammary carcinomas that have arisen spontaneously in strain C3H mice. We have established about 10 long-term strains which have been examined for the agent for quite a long time. In most of these the agent was maintained in culture for at least 6 months. Three of the strains were continued for longer than 3 years. One of these was tested again after 5 years, both the original strain and single cell clones. These tests were exhaustively made by Dr. Andervont on the susceptible C3Hf mouse, and no agent could be detected.

When the cells were implanted into mice, they produced cystic tumors, i.e., the clones, which were diagnosed by Dr. Dunn (National Institutes of Health) as anaplastic carcinoma. They were not differentiated carcinomas. The other two strains have been continued, and one of these reproduces the original tumor.

There is just slight evidence that a mammary-tumor agent is present. The data are almost too limited on the numbers of mammary tumors produced to be sure. These results, on carrying the mammary carcinoma in culture, resemble some of the data which Dr. Andervont and Dr. Dmochowski (M. D. Anderson Hospital) have collected on carrying some of these tumors for many passages in vivo in agent-free animals. In three of their studies, they have not been able to detect the agent after many transplants in the agent-free host, which again, of course, raises the question as to whether propagation of the virus is essential for continuation of the malignant properties of the cell.

I should like to ask Dr. Lasfargues whether he can strengthen up these points which Dr. Sabin raised with respect to the quantitative data on the propagation of the agent in the cultures. Now, he removes the used culture fluid, sediments the cells, and dilutes the supernatant fluid a 1000-fold to inoculate his mixed culture, as I understand his statements. I just wondered if there is a possibility that these supernatant fluids were not always cell-free. I wonder if he tried to filter the supernatant fluids to be absolutely sure, because it seems that, with this great dilution of material, he would have good evidence of propagation if he could be sure that these supernatant fluids did not contain cells.

An additional comment relates to the question of the role of the virus in the neoplastic change. We have isolated a single cell clone from normal parotid tissue of a strain C3H mouse and treated the cells in three separate experiments with the polyoma virus. We then implanted the cells, which were of C3H mouse origin, into hybrid mice and have obtained tumors in all three experiments with the treated cells but none from the controls. In order to be sure that these tumors were not induced in the hybrid host by virus, we have transplanted them and shown that they are specifically the C3H cells and not cells from the hybrid host. This is not closely related to your problem, but I did wonder if you had tried filtration of the supernatant fluids.

Dr. Dmochowski (M. D. Anderson Hospital): With respect to Dr. Dan Moore's statement earlier: Although it may be to him, and even to Dr. Bittner, a creed that the mammary agent is a virus, to some of us, at least, it is not a matter of creed but of scientific fact that the agent is a virus.