A quantitative study on the growth variability of tumour cell clones in vitro

Objectives: In this study, we quantify the growth variability of tumour cell clones from a human leukemia cell line. Materials and methods: We have used microplate spectrophotometry to measure the growth kinetics of hundreds of individual cell clones from the Molt3 cell line. The growth rate of each clonal population has been estimated by fitting experimental data with the logistic equation. Results: The growth rates were observed to vary among different clones. Up to six clones with a growth rate above or below the mean growth rate of the parent population were further cloned and the growth rates of their offsprings were measured. The distribution of the growth rates of the subclones did not significantly differ from that of the parent population thus suggesting that growth variability has an epigenetic origin. To explain the observed distributions of clonal growth rates we have developed a probabilistic model assuming that the fluctuations in the number of mitochondria through successive cell cycles are the leading cause of growth variability. For fitting purposes, we have estimated experimentally by flow cytometry the maximum average number of mitochondria in Molt3 cells. The model fits nicely the observed distributions of growth rates, however, cells in which the mitochondria were rendered non functional (rho-0 cells) showed only a 30% reduction in the clonal growth variability with respect to normal cells. Conclusions: A tumor cell population is a dynamic ensemble of clones with highly variable growth rate. At least part of this variability is due to fluctuations in the number of mitochondria.

💡 Research Summary

The authors set out to quantify the variability of growth rates among individual tumour cell clones derived from the human T‑lymphoblastoid cell line Molt3. Using a limiting‑dilution protocol they seeded approximately 0.3 cells per well into 96‑well plates, which, according to Poisson statistics, yields a large number of wells containing a single cell. Over the course of several days the growth of each well was monitored non‑invasively with a microplate spectrophotometer measuring absorbance at 730 nm. This wavelength is insensitive to the phenol‑red pH indicator in the medium, allowing the optical density increase to reflect cell number alone. Parallel validation with manual cell counting and ATP‑luminescence confirmed the reliability of the spectrophotometric read‑out.

Growth curves for each clonal population were fitted to the logistic equation
y(t)=y₀ + a/(1+exp