Assay of Frozen Mouse Mammary Carcinoma for the Mammary Tumor Milk Agent^*

Salvin-Moore, in 1908, determined that tumors would develop in mice following the subcutaneous implantation of neoplasms which had been exposed to the temperature of liquid air (−195° C.). He proposed a virus theory to explain the results, although he considered the possibility that some cells had remained viable.

In 1949, Gye and his associates submitted a similar theory based upon observations obtained following the freezing and drying of mouse cancers. Furthermore, Mann stated that when mammary cancers (both transplanted and spontaneous) were frozen, the milk agent virus was liberated in its “active” form and when injected subcutaneously, mammary tumors would be produced at the site of injection within a short interval of days or weeks. Few mammary tumors arose at other sites of inoculation because of the “selective infectivity” of the virus for mammary tissue.

To test for the active mammary tumor milk agent virus, transplanted mammary tumors were minced, suspended in dextrose, and frozen to −79° C. The thawed tumor-mince produced mammary tumors at the site of injection only in mice of stocks which would be susceptible to fresh tissue grafts of the tumor being tested. Negative results were obtained with mice of other strains, although they were susceptible to the development of spontaneous mammary cancer. Intraperitoneal inoculation of the frozen material was as effective as subcutaneous injection in the production of mammary tumors.

Whereas cell-free centrifugates have been found to contain the mammary tumor milk agent, cell-free extracts of the frozen mammary tumor-mince failed to induce tumors, within the specified interval, in any animal regardless of its genetic constitution.

Preliminary data indicate that the milk agent was no more active in cell-free extracts of the frozen than the fresh tissue of the same tumor, regardless of the route of administration. The final results of these tests will not be apparent for months, but the earliest tumors to be observed developed in mice which had received intraperitoneal injections.

The revival of this viral theory for mouse cancer by Gye et al. and Mann disregarded the previous finding of many investigators that mouse tissues (normal and cancerous) survived freezing to low temperatures. Evidence was also cited that the newly designed desiccator, used for their studies, did not destroy all cells. The fact that other methods of dehydration, including their own studies, had given negative results which would refute the virus hypothesis was disregarded as due to imperfect technic.

The experimental data submitted by Gye et al. and Mann were, without a doubt, based upon observations which they either incorrectly interpreted, as in the case of the studies with frozen tissues, or secured by using new equipment before it had been adequately tested and which resulted in imperfect technic.