Probing the compressibility of tumor cell nuclei by combined atomic force–confocal microscopy

Abstract

The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m(-1), force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

Mechanical Criterion for the Rupture of a Cell Membrane under Compression

“…For strong indentations, the indentation depth reaches a maximum value, Dz ¼ Dz max , corresponding to the cell becoming infinitely rigid when compared to the microindenter. Indeed, the cell will appear progressively stiffer as the indentation increases relative to the sample thickness (14), saturating at indentations of~60% of the cell height, where the cell becomes nearly infinitely rigid (13,38). Our experimental results indicate that the moderate indentation regime is valid up to Dz cutoff zDd cutoff z0:3 mm, corresponding to the initial region of the indentation curve that is well described by Hertz's contact theory (see Supporting Materials and Methods for details), whereas the maximum indentation is estimated to be on the order of Dz max z0:6 mm (see below).…”

Research progress in quantifying the mechanical properties of single living cells using atomic force microscopy

“…In recent years, researchers have investigated the cell mechanics by combining AFM with fluorescence microscopy or confocal microscopy [78][79][80][81]. Labernadie et al [78] investigated the stiffness of macrophage podosome by combining AFM with correlative fluorescence microscopy, revealing that podosomes harbor two types of overlapping periodic stiffness variations throughout the life span which depended on F-actin and myosin II activity.…”

“…Chahine et al [79] have systematically investigated the effect of age and cytoskeletal disruptors on the mechanical properties of chondrocytes by integrating AFM with confocal fluorescence microscopy, showing that intermediate filament integrity is a key to the nonlinear elastic properties of chondrocytes. Krause et al [80] have combined AFM with confocal fluorescence microscopy to investigate the bulk stiffness of cellular nuclei using sphere probe, clearly visualizing the cantilever-nuclear contact during the process of indentation. Yu et al [81] have built an integrated imaging system by combining stimulated emission depletion microscope and AFM, which can simultaneously obtain mechanical parameters and fluorescence super-resolution images.…”

Advances in the application of nanotechnology in the diagnosis and treatment of gastrointestinal tumors

“…A patent approach incorporating AFM with confocal microscopy was developed to detect bulk nuclear stiffness and to simultaneously visualize the cantilever-nucleus contact. Krause et al (21) used this method to identify nuclear compressibility prior to and following nuclear softening induced by the chromatin-decondensating agent trichostatin A, with the results indicating that this approach may be practical. The proposed tool may be extremely useful for the comprehension of the concept of limiting nuclear deformation in carcinoma invasion (21).…”

“…Krause et al (21) used this method to identify nuclear compressibility prior to and following nuclear softening induced by the chromatin-decondensating agent trichostatin A, with the results indicating that this approach may be practical. The proposed tool may be extremely useful for the comprehension of the concept of limiting nuclear deformation in carcinoma invasion (21). Wu et al (22) used a microplate measurement system approach based on the deflection of a flexible microcantilever to measure cell stiffness (in Pa) and adhesion force (in nN) between cells prior to and following transforming growth factor-β1-induced epithelial-to-mesenchymal transition.…”