Functional evaluation of circulating anti-cancer antibodies with a 3D tumor cell growth inhibition assay

Antibodies directed against surface antigens of tumor cells are commonly found in sera of cancer patients and of oncological animal models. Polyclonal antibodies directed against various epitopes of the same antigen may be spontaneously elicited by tumor antigens or may result from the administration of specific vaccines and other immunostimulating treatments. Furthermore, after therapeutic administration of monoclonal antibodies, the antibody will be detectable in the bloodstream for several weeks. Circulating antibodies are easily detected with enzyme-linked immunosorbent assays (ELISA) and other immunometric tests which, however, cannot tell whether the antibodies are functional, i.e. whether they can significantly inhibit (or enhance) tumor growth. One possibility would be to treat conventional (i.e. bi-dimensional, 2D) tumor cell cultures with antibody-containing sera. However, in several instances, it was found that 2D cultures were poorly sensitive, even to powerful monoclonal antibodies like trastuzumab, whereas three-dimensional (3D) cultures may better reveal the tumor-inhibitory activity of circulating antibodies. We describe here a breast cancer 3D soft agar colony growth inhibition assay that was developed to quantify the tumor cell inhibitory activity of antibodies against human HER-2 elicited in mice by specific vaccines. The assay might be readily modified to analyze antibodies against different surface antigens expressed by other tumor types and also for testing of new monoclonal antibodies and nanobodies.