Purified membrane fractions from mammary tumor cells elicit biological reactivity in in vitro cell-mediated immune reactions

Lithium chloride (LiCl) aggregation followed by centrifugation through a sucrose step gradient was used to obtain purified plasma membranes from two sublines of mouse mammary adenocarcinoma. These two tumor lines were chemically induced by treatment of a hyperplastic nodule with 7,12-dimethylbenzanthracene (DMBA). One line, D1-DMBA-3, has been found to be immunogenic to the host of origin, while the other, D1-DMBA-2, does not elicit specific tumor immunity as previously tested in in vivo and in vitro immune reactions. The purified membrane fractions were assayed for protein, DNA and sialic acid content as well as enzymatic markers of membrane purity. When tested in blastogenesis and cytotoxicity reactions, membrane-containing fractions from the D1-DMBA-3 immunogenic tumor were found to be stimulatory to spleen cells of D1-DMBA-3 tumor bearers over a ten-fold protein concentration. Spleen cells from normal mice do not respond in these reactions to the various tumor fractions. No reactivity was observed when non-membrane-containing preparations were used as stimuli in the cell-mediated immune reactions. The specificity of these reactivities was further demonstrated by the lack of responses when fractions from the non-immunogenic D1-DMBA-2 tumor were tested in parallel in our in vitro assays. The data presented indicate that the procedure employed is useful for the isolation of membrane-associated, tumor-specific antigens which can be easily quantitated and still retain biological activity in in vitro tests of cell-mediated immunity.