Successive B16 melanoma tumor lines were selected for their ability to form pulmonary tumor nodules. This was accomplished by injecting tumor cells i.v. into syngeneic C57BL/6 mice and 2 to 3 weeks later collecting the secondary tumor growths and placing them into tissue culture. These tumor cells were then injected i.v. into new syngeneic mice and the process was repeated several times. With each successive tumor line the number of experimental lung tumor nodules was significantly increased. The B16 lines were found to be stable in their metastatic properties even after many subculturings in vitro.

These studies demonstrated that tumor cells that succeed in forming pulmonary tumor colonies also had increased invasive properties into normal tissues when implanted s.c. In addition, the tumor cell lines were prelabeled in vitro with [¹²⁵I]-5-iodo-2′-deoxyuridine and suspensions of labeled cells were injected i.v. into normal syngeneic hosts. Animals were killed at intervals afterwards, and the lungs and blood were processed and monitored for radioactivity. At any time interval, the lungs of mice treated by injections of cells of high metastatic yield contained more tumor cells. The differences in tumor cell numbers in the lungs were most pronounced immediately following i.v. injection. These results suggest that an increased initial arrest of highly metastatic cells in a capillary bed may be a major factor in their increased survival.

Low numbers of normal lymphocytes or lymphocytes from syngeneic mice immunized to the B16 melanoma, when mixed in vitro with the tumor cells, lead to the formation of multitumor cell-lymphocyte clumps. The degree of clumping was related to both the type of the metastatic tumor line and/or the syngeneic lymphocyte.

These results support the hypothesis that the survival of invasive and/or circulating malignant tumor cells is not a random phenomenon; rather it appears that malignant cells possess unique qualities which allow for their survival.