Microrheological comparison of melanoma cells by atomic force microscopy

Abstract

Melanoma is one of the most severe cancers due to its great potential to form metastasis. Recent studies showed the importance of mechanical property assessment in metastasis formation which depends on the cytoskeleton dynamics and cell migration. Although cells are considered purely elastic, they are viscoelastic entities. Microrheology atomic force microscopy (AFM) enables the assessment of elasticity and viscous properties, which are relevant to cell behavior regulation. The current work compares the mechanical properties of human neonatal primary melanocytes (HNPMs) with two melanoma cell lines (WM793B and 1205LU cells), using microrheology AFM. Immunocytochemistry of F-actin filaments and phosphorylated focal adhesion kinase (p-FAK) and cell migration assays were performed to understand the differences found in microrheology AFM regarding the tumor cell lines tested. AFM revealed that HNPMs and tumor cell lines had distinct mechanical properties. HNPMs were softer, less viscous, presenting a higher power-law than melanoma cells. Immunostaining showed that metastatic 1205LU cells expressed more p-FAK than WM793B cells. Melanoma cell migration assays showed that WM73B did not close the gap, in contrast to 1205LU cells, which closed the gap at the end of 23 h. These data seem to corroborate the high migratory behavior of 1205LU cells. Microrheology AFM applied to HNPMs and melanoma cells allowed the quantification of elasticity, viscous properties, glassy phase, and power-law properties, which have an impact in cell migration and metastasis formation. AFM study is important since it can be used as a biomarker of the different stages of the disease in melanoma.

Keywords: AFM; Frequency sweep; Melanocytes; Melanoma; Microrheology atomic force microscopy; Power-law exponent; Viscoelasticity.

Fig. 1

Representative examples of indentation using AFM cantilever PFQNM-LC-A-CAL to quantify the mechanical properties of HNPMs, WM793B, and 1205LU cells. The tip pointed at the cell nucleus

Fig. 2

Histograms of mechanical properties quantification of HNPMs (red), WM793B (blue), and 1205LU (black) cells: a Storage modulus; and b loss structure damping modulus. The box plot above the histograms present the median (red line) and the 25th and 75th percentiles (extent of rectangle in cream color), and the 10th and 90th percentiles (black lines extending beyond the box)

Fig. 3

Histograms of mechanical properties quantification of HNPMs (red), WM793B (blue), and 1205LU (black) cells: a Power-law exponent of the storage modulus; and b loss tangent. The box plot above the histograms present the median (red line) and the 25th and 75th percentiles (extent of rectangle in cream color), and the 10th and 90th percentiles (black lines extending beyond the box)

Fig. 4

F-actin distribution in melanoma cell lines (WM793B and 1205LU). Staining of cells for DAPI to identify the cell nuclei (blue), p-FAK (red), and phalloidin from F-actin filaments (green). The overlap of the three channels is presented in the bottom Z-stack and the Z-maximum projection of the imaged area. Scale bars: 20 µm

Fig. 5

Cell migration assay of WM793B and 1205LU cell lines between 0 and 23 h. a WM793B cell line. b 1205LU cell line. c Gap size (i.e. state of closure) during the 23 h of the assay, for both cell lines