AFM stiffness nanotomography of normal, metaplastic and dysplastic human esophageal cells

Abstract

The mechanical stiffness of individual cells is important in tissue homeostasis, cell growth, division, and motility, and the epithelial-mesenchymal transition in the initiation of cancer. In this work, a normal squamous cell line (EPC2) and metaplastic (CP-A) as well as dysplastic (CP-D) Barrett’s Esophagus columnar cell lines are studied as a model of pre-neoplastic progression in the human esophagus. We used the combination of an atomic force microscope (AFM) with a scanning confocal fluorescence lifetime imaging microscope (FLIM) to study the mechanical properties of single adherent cells. 64 force indentation curves were taken over the nucleus of each cell in an 8×8 grid pattern. Analyzing the force indentation curves, indentation depth dependent Young’s moduli were found for all cell lines. Stiffness tomograms demonstrate distinct differences between the mechanical properties of the studied cell lines. Comparing the stiffness for indentation forces of 1 nN, most probable Young’s moduli were calculated to 4.7 kPa for EPC2 (n=18 cells), 3.1 kPa for CP-A (n=10), and 2.6 kPa for CP-D (n=19). We also tested the influence of nuclei and nucleoli staining organic dyes on the mechanical properties of the cells. For stained EPC2 cells (n=5), significant stiffening was found (9.9 kPa), while CP-A cells (n=5) showed no clear trend (2.9 kPa) and a slight softening was observed (2.1 kPa) in the case of CP-D cells (n=16). Some force-indentation curves show non-monotonic discontinuities with segments of negative slope, resembling a sawtooth pattern. We found the incidence of these ‘breakthrough events’ to be highest in the dysplastic CP-D cells, intermediate in the metaplastic CP-A cells, and lowest in the normal EPC2 cells. This observation suggests that the microscopic explanation for the increased compliance of cancerous and pre-cancerous cells may lie in their susceptibility to ‘crumble and yield’ rather than their ability to ‘bend and flex’.