A high throughput approach for analysis of cell nuclear deformability at single cell level

Abstract

Various physiological and pathological processes, such as cell differentiation, migration, attachment, and metastasis are highly dependent on nuclear elasticity. Nuclear morphology directly reflects the elasticity of the nucleus. We propose that quantification of changes in nuclear morphology on surfaces with defined topography will enable us to assess nuclear elasticity and deformability. Here, we used soft lithography techniques to produce 3 dimensional (3-D) cell culture substrates decorated with micron sized pillar structures of variable aspect ratios and dimensions to induce changes in cellular and nuclear morphology. We developed a high content image analysis algorithm to quantify changes in nuclear morphology at the single-cell level in response to physical cues from the 3-D culture substrate. We present that nuclear stiffness can be used as a physical parameter to evaluate cancer cells based on their lineage and in comparison to non-cancerous cells originating from the same tissue type. This methodology can be exploited for systematic study of mechanical characteristics of large cell populations complementing conventional tools such as atomic force microscopy and nanoindentation.

Actomyosin, vimentin and LINC complex pull on osteosarcoma nuclei to deform on micropillar topography

“…We have already shown nuclear deformation in a variety of cell types. (20)(21)(22)(23) Recently the nuclear deformability of human pluripotent stem cells during early germ layer specification was investigated using 3D micropillar array platforms and found to be lost during ectoderm differentiation.…”

Loss of Nuclear Deformability of Breast Cancer Cells by the Disruption of Actin Filaments

“…supporting the studies reporting that the softest cancer cells were also most deformed on the micropatterned surfaces. 10,13,38 After 48 h, nuclei of all cells were deformed somewhat more; circularity values were now 0.55, 0.41 and 0.37 for MCF10A, MCF7and MDAMB231, respectively. Results show that eventually, all the cells are deformed but the deformation was slower for benign (MCF10A) cells whereas nuclei of invasive malignant (MDAMB231) cell deformed at a much higher rate.…”

“…Micropillar arrays were designed at METU BIOMATEN and outsourced to be fabricated using photolithography. 13,15,16 The micropillars were in square prism shape, 8 μm tall with 4x4 μm 2 area and 4 μm interpillar gap. Using wafers as a template, polydimethylsiloxane (PDMS) molds were prepared with Sylgard 184 silicone prepolymer and Sylgard 184 curing agent (Dow Corning Company, UK) (10:1 w/w) and cured for 4 h at 70 °C.…”

“…Common shapes used in the fabrication of surface topographies are pores, gratings, wells, pits, cones, posts, pillars, grooves or meshlike structures that can be organized in a regular pattern. [12][13][14][15][16] Several studies have shown that many cell types react strongly to micro and nano features on substrate surfaces. [13][14][15][16][17][18][19] On such surfaces, changes have been reported in the cell behavior such as adhesion, 20,21 alignment, 21-23 morphology (cytoskeletal organization), 17,24 proliferation, 25 viability, 26 gene expression and differentiation.…”

“…[12][13][14][15][16] Several studies have shown that many cell types react strongly to micro and nano features on substrate surfaces. [13][14][15][16][17][18][19] On such surfaces, changes have been reported in the cell behavior such as adhesion, 20,21 alignment, 21-23 morphology (cytoskeletal organization), 17,24 proliferation, 25 viability, 26 gene expression and differentiation. 14,27,28 In this study, main hypothesis was that the difference in the stiffness of the nuclei of malignant and benign breast cancer cells can be used as a discriminating factor in cancer detection because of the substantial deformation of the nuclei of the malignant cells that could be quantified through the extent of deformation of the cell nuclei.…”