Our objective is to develop multi-functional nanotechnology-based anti-tumor drug delivery systems for improving the efficacy of treatments and reducing undesirable side effects. The essential part of this process is the development and validation of un-biased, quantitative analytical techniques. We employed a newly developed holographic imaging cytometry system HoloMonitor® M4 for label-free time-lapse cellular analysis (Phase Holographic Imaging, Sweden).In previous work we have demonstrated our ability to obtain quantitative, high content cellular feature data congruent to data obtained from traditional label-based systems. We applied a modified version of the Kolmogorov-Smirnov 2-sample test. The classic test takes control and test frequency distributions (histograms), and converts them to probability functions. The maximum vertical displacement between the two is reported as the D-value, to determine if the two distributions are significantly different. In modified versions of the test, it is a comparator; instead of a single value, we obtain histograms of the difference between the control and test normalized probability functions. These histograms are termed Brownian Bridges, where the end points are fixed at a value of zero, and the function is free to vary in between, in either up-going, down-going or mixed. Now, we report the development of a 4-sample Kolmogorov based method. Two modified K.S. tests are performed to compare distributions A to Ac and B to Bc. It is assumed that all the distributions have the same time basis, and each time point is processed sequentially. The resulting time point pairs are plotted as a vector in a two parameter scattergram, tracing the outlines of the probability distribution of the cell populations through time. Unlike, the Brownian Bridge histograms, here, the starting and the ending points are both at the same location (0,0), within the two dimensional scattergram, but are free to move in both positive and negative directions away from the origin. HeLa cells in a 12-well MatTek plate were adhered and treated with Paclitaxel (PCT) in concentrations from 0 to 40 nM in 5-fold increments, either pulsed for 4 hrs. followed by 48 hours of imaging or with continuous exposure for 48 hours of imaging. We obtained a variety of different plot types, including continuous vs. pulsed comparisons, inter dosage comparisons, as well as the ability to compare multiple features to each other. Of particular significance is the fact that in comparative dosage experiments over 10 nM, the pulsed and continuous probability zones are completely separated, reflecting live and dead cells, and allowing complex pharmacodynamic tracking.

Citation Format: Ed Luther, Livia Mendes, Jiayi Pan, Elena Holden, Vladimir Torchilin. Development of a 4-sample version of the Kolmogorov-Smirnov test for evaluating the temporal physiology of cells treated with test compounds in a label-free, high content, platform for quantitative analysis of adherent cell-culture models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4553. doi:10.1158/1538-7445.AM2017-4553