DETERMINING T(POT) IN HETEROGENEOUS SYSTEMS - A NEW APPROACH ILLUSTRATED WITH MULTICELL SPHEROIDS

Like many tumors, multicell spheroids from Chinese hamster V79-171b fibroblasts display karyotypic heterogeneity as they enlarge. This creates significant problems for analysis of univariate DNA content histograms of cells from spheroids, and even for bivariate bromodeoxyuridine (BrdUrd)/DNA flow histograms. While attempting to routinely measure spheroid cell kinetics with the latter approach, a simple alternative that overcomes many of the inherent problems emerged. The exit rate of BrdUrd-labelled cells from the S-phase compartment(s), determined using standard DNA analysis software, is used to define the duration of S-phase and ultimately the potential doubling time of the clonogenic cell population. This approach appears to offer several advantages over the conventional ''relative movement'' technique for heterogeneous samples, particularly since the subjective distinction between labelled/divided tetraploid G1 cells vs. labelled/undivided diploid G2 is no longer required. Further, the technique avoids some potential artifacts that might be expected for heterogeneous tumors where cell subpopulations proliferate with quite different cell kinetics.