A novel cell-stiffness-fingerprinting analysis by scanning atomic force microscopy: comparison of fibroblasts and diverse cancer cell lines

Abstract

Differing stimuli affect cell-stiffness while cancer metastasis further relates to cell-stiffness. Cell-stiffness determined by atomic Force Microscopy (AFM) has been limited by measurement over nuclei to avoid spurious substratum effects in thin cytoplasmic domains, and we sought to develop a more complete approach including cytoplasmic areas. 90 μm square fields were recorded from 10 sites of cultured Human Dermal Fibroblasts (HDF), and 3 sites each for melanoma (MM39, WM175, MeIRMu), osteosarcoma (SAOS-2, U2OS), and ovarian carcinoma (COLO316, PEO4) cell lines, each site providing 1,024 measurements as 32x32 square grids. Stiffness recorded below 0.8 μm height was occasionally influenced by substratum, so only stiffness recorded above 0.8 μm was analyzed, but all sites were included for height and volume analysis. COLO316 had the lowest cell height and volume, followed by HDF (p<0.0001), and then PEO4, SAOS-2, MeIRMu, WM175, U2OS, and MM39. HDF were more stiff than all other cells (p < 0.0001), while in descending order of stiffness were PEO4, COLO316, WM175, SAOS-2, U2OS, MM39, and MeIRMu (p < 0.02). Stiffness-fingerprints comprised scattergrams of stiffness values plotted against the height at which each stiffness value was recorded, and appeared unique for each cell type studied, although in most cases the overall form of fingerprints was similar, with maximum stiffness at low height measurements and a second lower peak occurring at high height levels. We suggest our stiffness-fingerprint analytical method provides a more nuanced description than previously reported, and will facilitate study of the stiffness response to cell stimulation.

Potential Hydrodynamic Cytoplasmic Transfer between Mammalian Cells: Cell-Projection Pumping

“…Co-cultures were on gelatin (0.1% in PBS) coated surfaces with Fibroblasts seeded from 1 to 2 x 10 4 cells per cm 2 into either 25cm 2 AFM culture plates (4), culture well coverslips for CLSM, or 35mm glass bottom dishes for holotomography, and allowed to adhere overnight before labelling.…”

Potential hydrodynamic cytoplasmic transfer between mammalian cells: Cell-projection pumping

“…By contrast, cell and organellar turn-over renders highly specific organellar or protein labels unreliable for detecting cumulative cytoplasmic transfer between cells (1). Also important, is that Fib have appreciably greater cell surface stiffness compared with MC (23). Further, punctate organellar labelling aids recognition of transfer events in time-lapse microscopy.…”

“…Paraformaldehyde fixed monolayers were stored in PBS at 4 o C for combined fluorescence-AFM recordings of randomly selected cells (23). An Asylum Research MFP-3D-BIO atomic force microscope coupled with a Zeis Axio Observer A1 fluorescence microscope was used.…”

“…A 1 µm AFM spherical polystyrene probe was used to record 16 x 16 points of force curves over 50 µm x 50 µm areas. Asylum Research, Software Version IX Young's modulus for each point by the Hertz model (23,25). Height maps, bright field and fluorescence images were compared to localize discrete AFM measurement points to individual cells, and stiffness fingerprints were prepared (23).…”

“…Both fibroblasts and SAOS-2 were designated as belonging to one of two groups, being 'high' or 'low' labelling from the opposing cell type. Median AFM stiffness was determined for individual cells, while stiffness fingerprints were also made of cells according to group to address sampling limitations as earlier described (23). Prism 6.0e software (GraphPad Software Inc, La Jolla, CA) was used for statistical analysis.…”