To determine if changes in anchorage independent growth induced by transmembrane domain mutations were mirrored by altered tumorigenicity, nude mice were injected subcutaneously in the upper flanks with 253T-NT cell lines expressing wild type or mutant CEACAM1-4S. Subcutaneous tumors were harvested from animals at three weeks after injection, a time point chosen by necessity because of the large tumor burden in animals injected with 253T-NT cells expressing tyrosine mutants. On average, tumors produced by cells expressing wild type CEACAM1-4S were 5.5-and 6.5-fold larger by weight than those generated by cells transfected, respectively, with the empty vector or the single G424L mutant (Figure 8 ). While cells expressing the wild type protein formed tumors comparable in weight to those formed by cells expressing the double G to L mutant, 253T-NT cells expressing CEACAM1-4S with single Y448V, double Y445V/ Y448V or quadruple G424L/G432L/Y445V/Y448V mutations produced tumors that were 1.9-, 1.25-, and 2.14-fold larger than those produced by cells transfected with the wild type protein (Figure 8 ). Indirect immunofluorescence analysis of frozen tumor sections, confirmed that tumor nodules formed by 253-NT cells transfected with either wild type or transmembrane domain mutants remained strongly positive for CEACAM1-4S (Figure 9 ).