Mechanical Way To Study Molecular Structure of Pericellular Layer.

Atomic force microscopy (AFM) has been used to study the mechanical properties of cells, in particular, malignant cells. Softening of various cancer cells compared to their nonmalignant counterparts has been reported for various cell types. However, in most AFM studies, the pericellular layer was ignored. As was shown, it could substantially change the measured cell rigidity and miss important information on the physical properties of the pericellular layer. Here we take into account the pericellular layer by using the brush model to do the AFM indentation study of bladder epithelial bladder nonmalignant (HCV29) and cancerous (TCCSUP) cells. It allows us to measure not only the quasistatic Young's modulus of the cell body but also the physical properties of the pericellular layer (the equilibrium length and grafting density). We found that the inner pericellular brush was longer for cancer cells, but its grafting density was similar to that found for nonmalignant cells. The outer brush was much shorter and less dense for cancer cells. Furthermore, we demonstrate a method to convert the obtained physical properties of the pericellular layer into biochemical language better known to the cell biology community. It is done by using heparinase I and neuraminidase enzymatic treatments that remove specific molecular parts of the pericellular layer. The presented here approach can also be used to decipher the molecular composition of not only pericellular but also other molecular layers.

Correlation of cell mechanics with the resistance to malignant transformation in naked mole rat fibroblasts.

Naked mole rats (NMRs) demonstrate exceptional longevity and resistance to cancer. Using a biochemical approach, it was previously shown that the treatment of mouse fibroblast cells with RasV12 oncogene and SV40 Large T antigen (viral oncoprotein) led to malignant transformations of cells. In contrast, NMR fibroblasts were resistant to malignant transformations upon this treatment. Here we demonstrate that atomic force microscopy (AFM) can provide information which is in agreement with the above finding, and further, adds unique information about the physical properties of cells that is impossible to obtain by other existing techniques. AFM indentation data were collected from individual cells and subsequently processed through the brush model to obtain information about the mechanics of the cell body (absolute values of the effective Young's moduli). Furthermore, information about the physical properties of the pericellular layer surrounding the cells was obtained. We found a statistically significant decrease in the rigidity of mouse cells after the treatment, whereas there was no significant change found in the rigidity of NMR cells upon the treatment. We also found that the treatment caused a substantial increase in a long part of the pericellular layer in NMR cells only (the long brush was defined as having a size of >10 microns). The mouse cells and smaller brush did not show statistically significant changes upon treatment. The observed change in cell mechanics is in agreement with the frequently observed decrease in cell rigidity during progression towards cancer. The change in the pericellular layer due to the malignant transformation of fibroblast cells has practically not been studied, though it was shown that the removal of part of the pericellular layer of NMR fibroblasts made the cells susceptible to malignant transformation. Although it is plausible to speculate that the observed increase in the long part of the brush layer of NMR cells might help cells to resist malignant transformations, the significance of the observed change in the pericellular layer is yet to be understood. As of now, we can conclude that changes in cell mechanics might be used as an indication of the resistance of NMR cells to malignant transformations.

Ultrabright Fluorescent Silica Nanoparticles for Multiplexed Detection.

Fluorescent tagging is a popular method in biomedical research. Using multiple taggants of different but resolvable fluorescent spectra simultaneously (multiplexing), it is possible to obtain more comprehensive and faster information about various biochemical reactions and diseases, for example, in the method of flow cytometry. Here we report on a first demonstration of the synthesis of ultrabright fluorescent silica nanoporous nanoparticles (Star-dots), which have a large number of complex fluorescence spectra suitable for multiplexed applications. The spectra are obtained via simple physical mixing of different commercially available fluorescent dyes in a synthesizing bath. The resulting particles contain dye molecules encapsulated inside of cylindrical nanochannels of the silica matrix. The distance between the dye molecules is sufficiently small to attain Forster resonance energy transfer (FRET) coupling within a portion of the encapsulated dye molecules. As a result, one can have particles of multiple spectra that can be excited with just one wavelength. We show this for the mixing of five, three, and two dyes. Furthermore, the dyes can be mixed inside of particles in different proportions. This brings another dimension in the complexity of the obtained spectra and makes the number of different resolvable spectra practically unlimited. We demonstrate that the spectra obtained by different mixing of just two dyes inside of each particle can be easily distinguished by using a linear decomposition method. As a practical example, the errors of demultiplexing are measured when sets of a hundred particles are used for tagging.