Effects of methotrexate on the viscoelastic properties of single cells probed by atomic force microscopy

Abstract

Methotrexate is a commonly used anti-cancer chemotherapy drug. Cellular mechanical properties are fundamental parameters that reflect the physiological state of a cell. However, so far the role of cellular mechanical properties in the actions of methotrexate is still unclear. In recent years, probing the behaviors of single cells with the use of atomic force microscopy (AFM) has contributed much to the field of cell biomechanics. In this work, with the use of AFM, the effects of methotrexate on the viscoelastic properties of four types of cells were quantitatively investigated. The inhibitory and cytotoxic effects of methotrexate on the proliferation of cells were observed by optical and fluorescence microscopy. AFM indenting was used to measure the changes of cellular viscoelastic properties (Young's modulus and relaxation time) by using both conical tip and spherical tip, quantitatively showing that the stimulation of methotrexate resulted in a significant decrease of both cellular Young's modulus and relaxation times. The morphological changes of cells induced by methotrexate were visualized by AFM imaging. The study improves our understanding of methotrexate action and offers a novel way to quantify drug actions at the single-cell level by measuring cellular viscoelastic properties, which may have potential impacts on developing label-free methods for drug evaluation.

Atomic force microscopy studies on cellular elastic and viscoelastic properties

“…The cellular relaxation time measured by AFM approach-reside-retract experiments is dependent on the loading force of the probe (Li et al, 2016b). Therefore, in order to make the results comparable, the experimental conditions of MCF-10A and MCF-7 cells are identical in the present study.…”

“…The latter result can be attributed to the diverse viscoelastic behaviors of cytoplasm, whose flow enables the relaxation process of cells after indentation by the AFM tip (Moeendarbary et al, 2013); cytoplasm comprises heterogeneous components such as actin filaments, intermediate filaments, microtubules, nucleus, and organelles (Kirmizis and Logothetidis, 2010). Hence, a two-order Maxwell model can match the experimental stress relaxation curves well (Li et al, 2016a;Li et al, 2016b). Figure 2 shows the statistical contrast of elastic and viscoelastic properties between MCF-10A cells and MCF-7 cells measured by AFM approach-reside-retract experiments.…”

Atomic Force Microscopy in Characterizing Cell Mechanics for Biomedical Applications: A Review

“…The results showed that Young's modulus alone failed to indicate the metastatic progression, but the combined biomechanical signatures of Young's modulus, adhesion, and calcium dynamics could effectively indicate the metastatic potential of prostate cancer cells, inspiring that performing studies on measuring multiple types of cell mechanics (e.g., Young's modulus, adhesion, relaxation time) and combining them with biological properties may help us to develop better biomarkers (or biomarker combination) for exactly indicating cell states and pathological changes. We have used AFM to simultaneously measure the elastic (Young's modulus) and viscoelastic properties (relaxation time) of cells [45], [59] by simultaneously recording force curves and stress relaxation curves during the AFM indentation process on cells via an oscilloscope linked to AFM, which facilitates us to acquire multiple mechanical parameters for characterizing cell behaviors. In order to examine the role of more types of cell mechanics besides Young's modulus, direct comparison studies on cancer cells and their normal counterparts, particularly on primary cells prepared from clinical cancer patients, are required.…”

“…where F is the applied loading force of the AFM probe, A 0 is the instantaneous (purely elastic) response, A i are the [45], [59]. It should be noted that the elastic and viscoelastic properties of cells measured by AFM is dependent on the loading rate of AFM probe and the cell positions being probed also influence the measured Young's modulus of cells [60].…”

基于<bold>AFM</bold>的细胞弹性及黏弹性研究

“…(弹性系数、针尖形状)、基底、环境温度、探针加载 速度、细胞探测部位、培养基等 [6,29] . 利用AFM测量 细胞松弛时间受到加载力大小的影响 [27] . 因此, 为了 [14,15,30] .…”