scholarly journals Sequential Measurements of Catalytic Activities of Multi-Drug-Resistance Transporters and Cytochrome P450 Enzymes by Cytometry of Reaction Rate Constant

2020 ◽  
Author(s):  
Vasilij Koshkin ◽  
Mariana Bleker de Oliveira ◽  
Sven Kochmann ◽  
Chun Peng ◽  
Sergey N. Krylov
Keyword(s):  
Drug Resistance ◽  
Cytochrome P450 ◽  
Rate Constant ◽  
Reaction Rate ◽  
Reaction Rate Constant ◽  
Population Heterogeneity ◽  
Multi Drug Resistance ◽  
Specific Substrate ◽  
Cytochrome P450 Enzymes ◽  
Sequential Measurements

ABSTRACTCytometry of reaction rate constant (CRRC) is an accurate and robust approach to characterize cell-population heterogeneity using rate constants of cellular processes for which kinetic mechanisms are known. We work on a CRRC-based method to develop predictors of tumor chemoresistance driven by two processes: drug extrusion by multi-drug-resistance (MDR) transporters and drug inactivation by cytochrome-P450 enzymes (CYP). Each of the two possess is studied with its specific substrate and the process activity is characterized by a corresponding unimolecular rate constant. Due to the incompatibility of MDR and CYP assays, MDR and CYP activities may be difficult to measure simultaneously suggesting that they may need to be measured sequentially. The sequential measurements may also impose a problem: the results of the second assay may be affected by artifacts exerted by the first assay. The goal of this work was to understand whether the cells have a memory of the first assay that significantly affects the results of the second assay. To achieve this goal, we compared CRRC results for two orders of sequential measurements: the MDR→CYP order in which MDR activity is measured before CYP activity and the CYP→MDR order in which CYP activity is measured before MDR activity. It was found that the results of the CYP assay were similar in both orders; on the contrary, the results of the MDR assay were significantly different. Our findings suggest that MDR and CYP activity can be studied sequentially provided that MDR activity is measured first and CYP activity second.

scholarly journals Assessment of Heterogeneity of Cytochrome P450 Activity in Cancer-Cell Population by Cytometry of Reaction Rate Constant is Robust to Variation in Substrate Concentration

2020 ◽  
Author(s):  
Mariana Bleker de Oliveira ◽  
Vasilij Koshkin ◽  
Christopher G. R. Perry ◽  
Sergey N. Krylov
Keyword(s):  
Cytochrome P450 ◽  
Single Cell ◽  
Cell Population ◽  
Rate Constant ◽  
Substrate Concentration ◽  
Reaction Rate ◽  
Reaction Rate Constant ◽  
Single Cell Level ◽  
Cell Level ◽  
Key Players

ABSTRACTEnzymes of the cytochrome P450 (CYP) family catalyze the metabolism of chemotherapeutic agents and are among the key players in primary and acquired chemoresistance of cancer. The activity of CYP is heterogeneous in tumor tissues, and the quantitative characteristics of this heterogeneity can be used to predict chemoresistance. Cytometry of reaction rate constant (CRRC) is a kinetic approach to assess cell population heterogeneity by measuring rates of processes at the single-cell level via time-lapse imaging. CRRC was shown to be an accurate and robust method for assessing the heterogeneity of drug-extrusion activity catalyzed by ABC transporters, which are also key players in cancer chemoresistance. We hypothesized that CRRC is also a reliable method for assessing the heterogeneity of CYP activity. Here, we evaluated the robustness of assessing the heterogeneity of CYP activity by CRRC with respect to controlled variation in the concentration of a CYP substrate by comparing CRRC with non-kinetic approaches. We found that changing the substrate concentration by 20% resulted only in minimal changes in the position, width, and asymmetry of the peak in the CRRC histogram, while these parameters varied greatly in the non-kinetic histograms. Moreover, the Kolmogorov-Smirnov statistical test showed that the distribution of the cell population in CRRC histograms was not significantly different; the result was opposite for non-kinetic histograms. In conclusion, we were able to demonstrate the robustness of CRRC with respect to changes in substrate concentration when evaluating CYP activity at the single-cell level.

scholarly journals Multi-Drug-Resistance in Drug-Naive and Drug-Exposed Ovarian Cancer Cell Lines Responds Differently to Cell Culture Dimensionality

2020 ◽  
Author(s):  
Vasilij Koshkin ◽  
Mariana Bleker de Oliveira ◽  
Chun Peng ◽  
Laurie Aiiles ◽  
Geoffrey Liu ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Drug Resistance ◽  
Rate Constant ◽  
Cell Number ◽  
Reaction Rate Constant ◽  
Multi Drug Resistance ◽  
Cell Clustering ◽  
Ovarian Cancer Cell Lines ◽  
Efflux Rate Constant ◽  
Drug Naïve

Does cell clustering influence intrinsic and acquired multi-drug resistance (MDR) differently? To address this question, we studied cultured monolayers (representing individual cells) and cultured spheroids (representing clusters) formed by drug-naïve (intrinsic MDR) and drug-exposed (acquired MDR) lines of ovarian cancer A2780 cells by cytometry of reaction rate constant (CRRC). MDR efflux was characterized by accurate and robust “cell number vs. MDR efflux rate constant (kMDR)” histograms. Both drug-naïve and drug-exposed monolayer cells presented unimodal histograms; the histogram of drug-exposed cells was shifted towards higher kMDR value suggesting greater MDR activity. Spheroids of drug-naïve cells presented a bimodal histogram indicating the presence of two subpopulations with different MDR activity. In contrast, spheroids of drug-exposed cells presented a unimodal histogram qualitatively similar to that of the monolayers of drug-exposed cells but with a moderate shift towards greater MDR activity. The observed greater effect of cell clustering on intrinsic than on acquired MDR can help guide the development of new therapeutic strategies targeting clusters of circulating tumor cells.

scholarly journals Measuring Reaction Rate Constant in Individual Cells to Facilitate Accurate Analysis of Cell-Population Heterogeneity

2019 ◽  
Author(s):  
Vasilij Koshkin ◽  
Sven Kochmann ◽  
Apinaya Sorupanathan ◽  
Chun Peng ◽  
Laurie E. Ailles ◽  
...  
Keyword(s):  
Cell Population ◽  
Rate Constant ◽  
Reaction Rate ◽  
Time Lapse ◽  
Kinetic Mechanism ◽  
Reaction Rate Constant ◽  
Population Heterogeneity ◽  
Accurate Analysis ◽  
Disease Biomarkers ◽  
Molecular Reaction

We propose Cytometry of Reaction Rate Constant (CRRC) for accurate analysis of cell-population heterogeneity with respect to a specific molecular reaction. Conceptually, in CRRC, the cells are loaded with a reaction substrate, and its conversion into a product is followed by time-lapse fluorescence microscopy at the single-cell level. A reaction rate constant is determined for every cell by using a known kinetic mechanism of the reaction, and a kinetic histogram “number of cells vs. the rate constant” is built. Finally, this histogram is used to determine parameters of reaction-based cell-population heterogeneity. Here, we studied a reaction of substrate extrusion from cells by ABC transporters. We proved that sizes of subpopulations with different extrusion rates could be accurately determined from the kinetic histogram, and this determination was not significantly affected by change in substrate concentration. We foresee that CRRC will facilitate the development of reliable disease biomarkers based on parameters of reaction-based cell-population heterogeneity.

Measuring Reaction Rate Constant in Individual Cells to Facilitate Accurate Analysis of Cell-Population Heterogeneity

2018 ◽  
Author(s):  
Vasilij Koshkin ◽  
Sven Kochmann ◽  
Apinaya Sorupanathan ◽  
Chun Peng ◽  
Laurie E. Ailles ◽  
...  
Keyword(s):  
Cell Population ◽  
Rate Constant ◽  
Reaction Rate ◽  
Time Lapse ◽  
Kinetic Mechanism ◽  
Reaction Rate Constant ◽  
Population Heterogeneity ◽  
Accurate Analysis ◽  
Disease Biomarkers ◽  
Molecular Reaction

We propose Cytometry of Reaction Rate Constant (CRRC) for accurate analysis of cell-population heterogeneity with respect to a specific molecular reaction. Conceptually, in CRRC, the cells are loaded with a reaction substrate, and its conversion into a product is followed by time-lapse fluorescence microscopy at the single-cell level. A reaction rate constant is determined for every cell by using a known kinetic mechanism of the reaction, and a kinetic histogram “number of cells vs. the rate constant” is built. Finally, this histogram is used to determine parameters of reaction-based cell-population heterogeneity. Here, we studied a reaction of substrate extrusion from cells by ABC transporters. We proved that sizes of subpopulations with different extrusion rates could be accurately determined from the kinetic histogram, and this determination was not significantly affected by change in substrate concentration. We foresee that CRRC will facilitate the development of reliable disease biomarkers based on parameters of reaction-based cell-population heterogeneity.

scholarly journals Spheroid-Based Approach to Assess Tissue Relevance of Analysis of Dispersed-Settled Tissue Cells by Cytometry of Reaction Rate Constant

2020 ◽  
Author(s):  
Sergey Krylov ◽  
Vasilij Koshkin ◽  
Mariana Bleker de Oliveira ◽  
Chun Peng ◽  
Laurie E. Ailles ◽  
...  
Keyword(s):  
Rate Constant ◽  
Reaction Rate ◽  
Cell Model ◽  
Tissue Sample ◽  
Reaction Rate Constant ◽  
Cell Subpopulation ◽  
Multi Drug Resistance ◽  
Support Surface ◽  
Multicellular Spheroids ◽  
Fluorescent Substrate

File main pdf file (CRRC of dispersed-settled spheroidal cells.pdf) describes experimental results and their interpretation for a study of tissue relevance of analyses of dispersed-settled tissues cells by Cytometry of Reaction Rate Constant (CRRC). CRRC uses time-lapse fluorescence microscopy to measure a rate constant of a catalytic reaction in individual cells and, thus, facilitate accurate size determination for cell subpopulations with distinct efficiencies of this reaction. Practical CRRC requires that a tissue sample be disintegrated into a suspension of dispersed cells and these cells settle on the support surface before being analyzed by CRRC. We call such cells “dispersed-settled” to distinguish them from cells cultured as a monolayer. Studies of the dispersed-settled cells can be tissue-relevant only if the cells maintain their 3D tissue state during the multi-hour CRRC procedure. Here we propose an approach for assessing tissue relevance of the CRRC-based analysis of the dispersed-settled cells. Our approach utilizes cultured multicellular spheroids as a 3D cell model and cultured cell monolayers as a 2D cell model. The CRRC results of the dispersed-settled cells derived from spheroids are compared to those of spheroids and monolayers in order to find if the dispersed-settled cells are representative of the spheroids. To demonstrate its practical use, we applied this approach to a cellular reaction of multi-drug-resistance (MRD) transport which was followed by extrusion of a fluorescent substrate from the cells. The approach proved to be reliable and revealed long-term maintenance of MDR transport in the dispersed-settled cells obtained from cultured ovarian cancer spheroids. Accordingly, CRRC can be used to determine accurately the size of a cell subpopulation with an elevated level of MDR transport in tumor samples, which makes CRRC a suitable method for the development of MDR-based predictors of chemoresistance. The proposed spheroid-based approach for validation of CRRC is applicable to other types of cellular reactions, and, thus, will be an indispensable tool for transforming CRRC from an experimental technique into practical analytical tool. <div>Additional (zip) files contain supporting images, kinetic traces, and histograms. Their detailed descriptions are provided in the main pdf file. </div>

Measuring Reaction Rate Constant in Individual Cells to Facilitate Accurate Analysis of Cell-Population Heterogeneity

2019 ◽  
Author(s):  
Vasilij Koshkin ◽  
Sven Kochmann ◽  
Apinaya Sorupanathan ◽  
Chun Peng ◽  
Laurie E. Ailles ◽  
...  
Keyword(s):  
Cell Population ◽  
Rate Constant ◽  
Reaction Rate ◽  
Time Lapse ◽  
Kinetic Mechanism ◽  
Reaction Rate Constant ◽  
Population Heterogeneity ◽  
Accurate Analysis ◽  
Disease Biomarkers ◽  
Molecular Reaction

We propose Cytometry of Reaction Rate Constant (CRRC) for accurate analysis of cell-population heterogeneity with respect to a specific molecular reaction. Conceptually, in CRRC, the cells are loaded with a reaction substrate, and its conversion into a product is followed by time-lapse fluorescence microscopy at the single-cell level. A reaction rate constant is determined for every cell by using a known kinetic mechanism of the reaction, and a kinetic histogram “number of cells vs. the rate constant” is built. Finally, this histogram is used to determine parameters of reaction-based cell-population heterogeneity. Here, we studied a reaction of substrate extrusion from cells by ABC transporters. We proved that sizes of subpopulations with different extrusion rates could be accurately determined from the kinetic histogram, and this determination was not significantly affected by change in substrate concentration. We foresee that CRRC will facilitate the development of reliable disease biomarkers based on parameters of reaction-based cell-population heterogeneity.

scholarly journals Spheroid-Based Approach to Assess Tissue Relevance of Analysis of Dispersed-Settled Tissue Cells by Cytometry of Reaction Rate Constant

2020 ◽  
Author(s):  
Sergey Krylov ◽  
Vasilij Koshkin ◽  
Mariana Bleker de Oliveira ◽  
Chun Peng ◽  
Laurie E. Ailles ◽  
...  
Keyword(s):  
Rate Constant ◽  
Reaction Rate ◽  
Cell Model ◽  
Tissue Sample ◽  
Reaction Rate Constant ◽  
Cell Subpopulation ◽  
Multi Drug Resistance ◽  
Support Surface ◽  
Multicellular Spheroids ◽  
Fluorescent Substrate

File main pdf file (CRRC of dispersed-settled spheroidal cells.pdf) describes experimental results and their interpretation for a study of tissue relevance of analyses of dispersed-settled tissues cells by Cytometry of Reaction Rate Constant (CRRC). CRRC uses time-lapse fluorescence microscopy to measure a rate constant of a catalytic reaction in individual cells and, thus, facilitate accurate size determination for cell subpopulations with distinct efficiencies of this reaction. Practical CRRC requires that a tissue sample be disintegrated into a suspension of dispersed cells and these cells settle on the support surface before being analyzed by CRRC. We call such cells “dispersed-settled” to distinguish them from cells cultured as a monolayer. Studies of the dispersed-settled cells can be tissue-relevant only if the cells maintain their 3D tissue state during the multi-hour CRRC procedure. Here we propose an approach for assessing tissue relevance of the CRRC-based analysis of the dispersed-settled cells. Our approach utilizes cultured multicellular spheroids as a 3D cell model and cultured cell monolayers as a 2D cell model. The CRRC results of the dispersed-settled cells derived from spheroids are compared to those of spheroids and monolayers in order to find if the dispersed-settled cells are representative of the spheroids. To demonstrate its practical use, we applied this approach to a cellular reaction of multi-drug-resistance (MRD) transport which was followed by extrusion of a fluorescent substrate from the cells. The approach proved to be reliable and revealed long-term maintenance of MDR transport in the dispersed-settled cells obtained from cultured ovarian cancer spheroids. Accordingly, CRRC can be used to determine accurately the size of a cell subpopulation with an elevated level of MDR transport in tumor samples, which makes CRRC a suitable method for the development of MDR-based predictors of chemoresistance. The proposed spheroid-based approach for validation of CRRC is applicable to other types of cellular reactions, and, thus, will be an indispensable tool for transforming CRRC from an experimental technique into practical analytical tool. <div>Additional (zip) files contain supporting images, kinetic traces, and histograms. Their detailed descriptions are provided in the main pdf file. </div>

scholarly journals Multi-Drug-Resistance in Drug-Naive and Drug-Exposed Ovarian Cancer Cell Lines Responds Differently to Cell Culture Dimensionality

2020 ◽  
Author(s):  
Vasilij Koshkin ◽  
Mariana Bleker de Oliveira ◽  
Chun Peng ◽  
Laurie E. Ailles ◽  
Geoffrey Liu ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Drug Resistance ◽  
Rate Constant ◽  
Cell Number ◽  
Reaction Rate Constant ◽  
Multi Drug Resistance ◽  
Cell Clustering ◽  
Ovarian Cancer Cell Lines ◽  
Efflux Rate Constant ◽  
Drug Naïve

AbstractDoes cell clustering influence intrinsic and acquired multi-drug resistance (MDR) differently? To address this question, we studied cultured monolayers (representing individual cells) and cultured spheroids (representing clusters) formed by drug-naïve (intrinsic MDR) and drug-exposed (acquired MDR) lines of ovarian cancer A2780 cells by cytometry of reaction rate constant (CRRC). MDR efflux was characterized by accurate and robust “cell number vs. MDR efflux rate constant (kMDR)” histograms. Both drug-naïve and drug-exposed monolayer cells presented unimodal histograms; the histogram of drug-exposed cells was shifted towards higher kMDR value suggesting greater MDR activity. Spheroids of drug-naïve cells presented a bimodal histogram indicating the presence of two subpopulations with different MDR activity. In contrast, spheroids of drug-exposed cells presented a unimodal histogram qualitatively similar to that of the monolayers of drug-exposed cells but with a moderate shift towards greater MDR activity. The observed greater effect of cell clustering on intrinsic than on acquired MDR can help guide the development of new therapeutic strategies targeting clusters of circulating tumor cells.

scholarly journals Determination of exothermic batch reactor specific model parameters

2019 ◽  
Vol 292 ◽  
pp. 01063
Author(s):  
Lubomír Macků
Keyword(s):  
Rate Constant ◽  
Reaction Rate ◽  
Batch Reactor ◽  
Specific Model ◽  
Reaction Rate Constant ◽  
Order Reaction ◽  
First Order Reaction ◽  
Model Parameters ◽  
Reactor Model ◽  
First Order

An alternative method of determining exothermic reactor model parameters which include first order reaction rate constant is described in this paper. The method is based on known in reactor temperature development and is suitable for processes with changing quality of input substances. This method allows us to evaluate the reaction substances composition change and is also capable of the reaction rate constant (parameters of the Arrhenius equation) determination. Method can be used in exothermic batch or semi- batch reactors running processes based on the first order reaction. An example of such process is given here and the problem is shown on its mathematical model with the help of simulations.

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