Enzymes 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.

中文翻译：

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通过反应速率常数的细胞计数法评估癌细胞群体中细胞色素P450活性的异质性对底物浓度的变化是稳健的

细胞色素P450（CYP）家族的酶催化化学治疗剂的代谢，并且是癌症原发性和获得性化学耐药性的关键参与者。CYP的活性在肿瘤组织中是异质的，这种异质性的定量特征可用于预测化学耐药性。反应速率常数（CRRC）细胞计数法是一种动力学方法，通过通过延时成像在单细胞水平上测量过程速率来评估细胞群体异质性。研究表明，CRRC是评估ABC转运蛋白催化的药物挤出活性异质性的准确而可靠的方法，ABC转运蛋白也是癌症化学耐药性的关键参与者。我们假设CRRC也是评估CYP活性异质性的可靠方法。这里，我们通过将CRRC与非动力学方法进行比较，评估了通过CRRC评估CYP活性异质性相对于CYP底物浓度的受控变化的鲁棒性。我们发现将底物浓度改变20％只会导致CRRC直方图中峰的位置，宽度和峰的不对称性发生最小的变化，而这些参数在非动力学直方图中变化很大。此外，Kolmogorov-Smirnov统计检验表明，CRRC直方图中细胞群的分布没有显着差异；对于非运动直方图，结果相反。总之，当评估单细胞水平的CYP活性时，我们能够证明CRRC相对于底物浓度变化的鲁棒性。