Colonies formed in agar from human breast cancer and their identification as T-lymphocytes

Single cell suspensions prepared from human breast cancer specimens by collagenase digestion were cultured in soft agar with phytohemagglutinin-stimulated human lymphocyte-conditioned medium (PHA-LCM). In 6 of 10 different tumors, PHA-LCM-dependent clonal growth was develop. After 12-14 days of incubation, two morphologic types of colony containing 20-500 cells were recognized. Both were composed of lymphocytes of T-cell nature, as judged by cell morphology in smears, cytochemical properties, capacity to form rosettes with sheep erythrocytes, and electron microscopic appearances. Contamination of the tumor cell suspensions by blood could be excluded as a source of the colony-forming lymphocytes, and the incidence of colony-forming cells correlated well with the degree of lymphocyte infiltration of the tumors. Some of the colonies in agar were expanded further in liquid culture in the continuous presence of PHA-LCM. These clones were apparently high in proliferating capacity as compared with the proliferating activity of peripheral T-cell clones obtained from normal blood. These clones were considered to be highly activated T-lymphocytes and to be stimulated to grow in vitro by the T-cell growth factor contained in PHA-LCM. The direct cloning and expansion of such activated T-lymphocytes infiltrating the tumors will be useful for studies on the functional characteristics of these cells.