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

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.

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

10.1101/2020.07.12.199125 ◽

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.

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

10.1101/2020.10.15.340976 ◽

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.

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