The plasma membrane is believed to play an important role in the successful establishment of hematogenous metastases. The membrane bears (1) enzymes required for destruction of basement membrane barriers, (2) receptors for homotypic and heterotypic cell associations and (3) antigens which direct specific and nonspecific immune reactivities. I have used noncytolytic extraction with 1-butanol to enhance the incidence of experimentally induced pulmonary metastases. Butanol increases the metastatic potential of sublines of either low or high metastatic phenotype derived from the chemically induced fibrosarcoma, MCA-F, or the spontaneous melanoma, B16. Brief incubation of extracted cells in 1 to 10 micrograms of crude butanol extract (CBE) reconstituted the metastatic phenotype to that of the unextracted controls. This study will isolate and characterize the molecules which, when they are borne on the cell surface, reduce the potential of neoplastic cells to form hematogenous pulmonary metastases. The reconstitutive activity in CBE will be isolated, using a combination of preparative isoelectric focusing, gel filtration and lectin affinity chromatography and high-pressure liquid chromatography. The reconstitutive activity in partially purified preparations will be quantitatively assessed as the milligram amount of protein necessary to reduce the incidence of experimentally induced pulmonary metastases by 50%. Butanol extraction of target cells, but not effector cells, reduces the percent natural cell-mediated cytotoxicity (NCMC) in a 4-hour ?51?Cr-release assay. Crude and partially purified extracts will be used either in soluble form or associated with target cells to determine whether the membrane moieties responsible for the modulation of hematogenous metastases serve as target structures for NCMC. Immunization of syngeneic hosts with CBE before intravenous challenge significantly increases the incidence of experimentally induced lung metastases, and this effect can be adoptively transferred with spleen cells. Interestingly, transfer of spleen cells can be made either before or after challenge, suggesting that CBE elicits an immune response which specifically potentiates the growth of tumor in the lungs. The final objective of this project is to delineate the cellular immune response to antigens in CBE and to characterize the antigens and immune cells participating. Thus, butanol extraction of metastatic cells will allow investigation of possible mechanisms involved in hematogenous metastasis, of the role of tumor cell surface structures in metastasis and of the effect of antigen stimulation on the eventual development of metastases.
