Abstract A59: Treatment with targeted anticancer agent affects rapidly growing subclones: An integrated in vitro/in silico analysis of clonal heterogeneity in soft agar.
Authors: Zopf Christopher J. , Szynal Carmin , Palani Santhosh , Bowman Doug , Bradley John , Mettetal Jerome , Ecsedy Jeffrey , Dorner Andy , Shyu Wen Chyi , Kuida Keisuke , Blair Derek , Chakravarty Arijit 
Published: August 2014

Abstract
A growing body of clinical evidence supports a view that the often observed refractory tumor rebound arises from variation in treatment response among tumor subclones already present in the tumor at the time of diagnosis. Heterogeneity within the tumor provides subpopulations of varying fitness under the selective pressure of treatment. Through evolutionary dynamics, sensitive subpopulations wither under purifying selection, while resistant subclones replenish the population.
In this report, we employ a previously developed in vitro system based on the soft agar clonogenic assay to characterize the degree of heterogeneity in cell growth rate (fitness) between subclones in a population. The system consists of high-throughput incubation and daily imaging of colonies growing in wells of titrated treatment conditions over two to three weeks, followed by automated image analysis, image registration, and colony tracking. Individual colony growth kinetics are well-described by a single-exponential model with fit growth rates uncorrelated to location within the well. Growth rate is likely inherited over the course of the experiment as seen by a strong correlation to final diameter, and further supported by Gillespie stochastic simulations showing the final, untreated population growth rate distribution could only be achieved if heritability was strong.
The methods described here are then applied to HCT-116 colonies allowed to grow for a week and then treated with targeted anticancer agents (in the PI-3K and MAP kinase pathways). This allowed direct comparison of individual colony growth rates pre- and post-treatment. We find growth rate scales down proportionally after treatment as faster growing colonies experience a greater decrease in growth rate. Comparing the post-treatment growth rates of the slowest and fastest growing colonies demonstrates the majority of projected population debulking occurs by inhibiting the fastest growing subclones. Scatter plots of post- vs. pre-treatment growth rate show the effect of treatment is somewhat stochastic: not all fast-growing colonies are affected to the same extent.
Heterogeneity in the population, then, serves to increase the likelihood of the existence of a fast-growing subclone with greater resistance to treatment. This enables the population to more rapidly and assuredly evolve through selection on pre-existing variation in subclones. Thus, natural selection in response to treatment, rather than biochemical or mutational adaptation, appears to drive the emergence of resistance in the population. Because of the high degree of heterogeneity in subclonal growth rates, this resistance can emerge rapidly in soft agar (over several days). The methodology described may be applied to gain insight into heterogeneity in cancer cell line treatment response for drugs with various mechanisms of action.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A59.
Citation Format: Christopher J. Zopf, Carmin Szynal, Santhosh Palani, Doug Bowman, John Bradley, Jerome Mettetal, Jeffrey Ecsedy, Andy Dorner, Wen Chyi Shyu, Keisuke Kuida, Derek Blair, Arijit Chakravarty. Treatment with targeted anticancer agent affects rapidly growing subclones: An integrated in vitro/in silico analysis of clonal heterogeneity in soft agar. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A59.