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• W2594659562 abstract "Our objective is to develop multi-functional nanotechnology-based anti-tumor drug delivery systems for improving efficacy of treatments and reducing undesirable side effects. The essential part of this process is the development of un-biased quantitative analytical techniques. We are reporting a successful validation of a very high content, medium throughput system in multi-well plates. We employed a newly developed holographic imaging cytometry system HoloMonitor® M4 for label-free time-lapse cellular analysis (Phase Holographic Imaging, Sweden), typically at 5 minute intervals for 48–72 hours. A low power red laser is split into sample and reference beams to obtain holograms of cells. The holograms are unwrapped by proprietary software into quantitative dark field images, with very precise calculation of the cell optical thickness. These images are segmented, and a vast variety of features are extracted for cellular events. In our evaluations we found that cell optical thickness, volume and area have high correlation with features used in traditional fluorescent DNA-stained analysis. We obtain full cell cycle profiles, including the separation of mitotic cells, cells undergoing mitotic dysfunction, and apoptotic cells—all in label-free environment. We recently developed a novel 4D image display of evaluated fields of view: X position, Y position, cell thickness coded as brightness over time in the Z direction. In these images, the history of the viewing area over time is displayed, and salient features such as cell proliferation, cell thickness, and cell motility, contact inhibition, and cell death are discernable. As an example, a combinatorial liposomal formulation containing paclitaxel and a P-gp inhibitor tariquidar was compared to free paclitaxel in SKOV3 TR (taxol resistant) cells seeded in Petri dishes. TR cells treated with free paclitaxel presented increased mitoses, while TR cells treated with the combinatorial preparation exhibited a complete abrogation of mitotic division. Cells were frozen in mitosis due to the polymerization of tubulin, the mechanism of paclitaxel toxicity. In this study Hela cells were seeded in a 6-well plate and allowed to acclimate overnight. Free doxorubicin (DOX) was added to 5 wells at concentrations ranging from 1.6 nM to 1 uM in 5-fold increments. Three fields (0.56 mm2) in each well were imaged for 48 hours at 5 min. intervals. We developed a modified version of the Kolmogorov-Smirnov 2-sample test to analyze the data. 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, which quantifies the amount of difference between the two. In our version, we obtain histograms of the D-values. We set up our analysis so the X-axis is time, and the feature is any of the reported metrics from the HoloMonitorM4 software. Our results are briefly summarized in the following table indicating where the sample is greater than the control (+), less than the control (-), and a transition (/). Dox Con......(1.6 nM).(8 nM).....(40 nM)..(200 nM)..(1 uM) Cell Count.........(+).......(-)........(---).......(---).......(---) Area (-)......(+)....(+++++)..(++/-)...(++/-) Max. Thick.........(+)....(++)...(+++++)...(-/+)......(-/+) Volume (+)......(+)....(+++++)...(++)......(++) Perimeter........ (+/-)...(+/-).......(++)....(--/++)...(-/++) Roughness.........(-).......(-).......(++/-)....(++/-)......(--) The results are consistent with the known effects of Dox. At low concentration, unrepaired DNA strand breaks start to accumulate and cause slight increases in the volume and thickness of the cells. At mid-concentration the DNA repair process is overwhelmed, proliferation is halted, and vast changes in cellular morphology are evident. At the higher concentration, changes in morphology correspond with cell death followed by eventual deterioration. Citation Format: Ed Luther, Livia Mendes, Daniel Costa, Jiayi Pan, Elena Holden, Vladimir Torchilin. A label-free, high content, moderate throughput analytical platform for quantitative kinetic analysis of cell behavior upon drug activation in cell-culture models based on the Kolmogorov-Smirnov test. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B18." @default.
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• W2594659562 date "2017-01-15" @default.
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• W2594659562 title "Abstract B18: A label-free, high content, moderate throughput analytical platform for quantitative kinetic analysis of cell behavior upon drug activation in cell-culture models based on the Kolmogorov-Smirnov test" @default.
• W2594659562 doi "https://doi.org/10.1158/1538-7445.epso16-b18" @default.
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