Cell membrane topology analysis by RICM enables marker-free adhesion strength quantification

Abstract

Reflection interference contrast microscopy (RICM) allows the visualization of the cell's adhesion topology on substrates. Here it is applied as a new label-free method to measure adhesion forces between tumor cells and their substrate without any external manipulation, i.e., the application of force or adjustments in the substrate elasticity. Malignant cancer transformation is closely associated with the down-regulation of adhesion proteins and the consequent reduction of adhesion forces. By analyzing the size and distribution of adhesion patches from a benign and a malignant human pancreatic tumor cell line, we established a model for calculating the adhesion strength based on RICM images. Further, we could show that the cell's spread area does not necessarily scale with adhesion strength. Despite the larger projected cell area of the malignant cell line, adhesion strength was clearly reduced. This underscores the importance of adhesion patch analysis. The calculated force values were verified by microfluidic detachment assays. Static and dynamic RICM measurements produce numerous adhesion-related parameters from which characteristic cell signatures can be derived. Such a cellular fingerprint can refine the process of categorizing cell lines according to their grade of differentiation.

Characterizing Cell Adhesion by Using Micropipette Aspiration

“…Indeed, these adhesive areas appear as dark patches in IRM images taken in the in-plane mode (18,22,23). By monitoring the evolution of these adhesive areas over the duration of a complete detachment event, we found that the detachment does not occur by peeling from the periphery but rather appears to be initiated in the region directly under the micropipette, see Movie S2 and Fig.…”

“…(B) Plot of the experimental intensity extrema positions versus the predictions from Eq. 1 (18,22), for l ¼ 340 nm. The good fit for this value of l is evidenced by the slope of the linear regression: 1.003 5 0.007.…”

“…1 A, inset). We measured the positions of the local intensity extrema as a function of h and verified that they follow the established equation (18,22) …”

“…We can perform detachment assays on a number of individual cells in a single experimental run, thus exploring a range of cell sizes, while imposing the micropipette diameter and the aspirating pressure rate (or loading rate). Finally, interference reflection microscopy (IRM) is used to monitor the adhesion areas, as was previously done in the case of adhering blood cells (9) and tumor cells (18). In a nutshell, this technique enables us to fully probe the influence of geometric parameters, such as cell surface, adhesion areas, and micropipette size, as well as dynamical parameters such as the loading rate.…”

“…Previous techniques at the single-cell level relied on pulling a micro-or nanoneedle inserted into the cell body (6)(7)(8), pulling (9,10) or scraping (11,12) the cell of interest with a bending cantilever, and aspirating the cell with a micropipette (13)(14)(15)(16). Experiments were also carried out on cell populations in which cell detachment was induced by centrifugation or hydrodynamic stress (17,18), yielding ensemble-averaged values for the quantities of interest. However, some of these studies involve technical difficulties, such as the need for protein scaffolding of the cells to avoid membrane rupture when they are pulled by a nanoneedle (8).…”