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’.

Cell stiffness determined by atomic force microscopy and its correlation with cell motility

“…Thus, due to the differences in microenvironments, cells derived from normal tissues and tumors are intrinsically different in their stiffness, which allows Young's modulus to be a reliable biomarker in the evaluation of metastasis and the motility of cells. In addition, the morphological observations support this opinion experimentally, showing that the cancerous cells usually have decreased stress fibers [25,29,33,67,68], increased cortical actin [25,33], and more lamellipodia [26,65], which benefit the invasion of cells by decreasing adhesion with the substrate and increasing the contraction forces for deformation and protrusion [27,64]. Taking into account that the nuclei are usually softer in cells with higher motility [58] and that the cortex actin network is much softer than the stress fibers [41], it is reasonable to detect a lower Young's modulus in metastatic carcinoma cells.…”

AFM detection of biophysical characteristics of specific regulatory T Cells after allogeneic hematopoietic stem cell transplantation

“…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 [22]. In this study, we found that the rigidity of Treg cells from patients with GVHD were similar to Treg cells without GVHD by the detection of adhesion force.…”

Ultra-rapid non-invasive clinical nano-diagnostic of inflammatory diseases

“…This study may lunch further mechanism understandings and optimizations for prognosis and prevention of any blood-related disorders, particularly on diagnosis and profiling the inflammatory modifications at different degrees level. Beyond imaging, AFM provides a "real time" study of living organisms, emerging multifunctional application tools such as: morphology, molecular forces 19,20 and mechanical properties 32,33 . However, AFM requires the immobilization of the cells on bare surfaces, which may influence the cell morphology 34 .…”

“…By isolating, immobilizing and washing the cells in one step, we significantly shorten the time for sample preparation, which does not exceed 5 minutes. Even the following measurements on AFM can require longer time and expertise at the beginning, for diagnostic purpose a fully automatic system to analyse cell 21 can be combined and developed 32,33 . Currently, the clinical diagnostic is based on the morphology of the cells, stained with specific antibodies, using conventional microscopic techniques.…”