Isolation, Karyotype, and Clonal Growth of Heterogeneous Subpopulations of Human Malignant Gliomas¹

The histological pleomorphism of human malignant gliomas as well as their variable response to therapy suggests that such tumors are not homogeneous but are composed of a heterogeneous population of cells. To search for heterogeneity in these tumors, we developed a protocol to identify by karyotype, isolate, and clone the subpopulations of the tumor. Freshly resected tumors were mechanically dissociated into single cells that were grown in suspension and short-term monolayer cultures. Chromosomal preparations were collected over the first 6 to 72 hr postresection. The karyotypes prepared comprised the “reference set” of chromosomes found in each tumor. The dissociated single cells were also dilution plated for monolayer culture. Cells that attached were marked and isolated as clones whose karyotypes were compared to those in the reference set in order to identify those clones that were cellular representatives of the tumor. Explant cultures established from the same tumor were grown in monolayer culture and cloned; their karyotypes were likewise compared to those in the reference set. Eight gliomas were analyzed. Each had metaphases that ranged in chromosome number from hypodiploid to hyperploid, but the frequency distribution of the subpopulations differed among the tumors. Defining a cellular subpopulation in the tumor as one yielding at least five karyotypically identical cells in the reference set, we found that each tumor contained from three to 21 subpopulations. All but one tumor produced 100 or more clones, and in seven tumors, more than 50% of the clones could be expanded for karyotyping; 7.6 to 25% of these clones had karyotypes identifiable in the reference set of their tumor. There was little relationship between the morphology of the clones and their karyotypes or growth kinetics. While explant cultures grew well, no clone derived from such cultures could be identified in the reference set. The protocol permits studies of clonal lines identifiable as cellular representatives of a patient's tumor and not variants generated in tissue culture. These studies confirmed that human malignant gliomas are comprised of karyotypically heterogeneous cellular subpopulations. Preliminary work suggests that their phenotypic behavior may be similarly heterogeneous.