Cytoskeletal stiffness, friction, and fluidity of cancer cell lines with different metastatic potential.

We quantified mechanical properties of cancer cells differing in metastatic potential. These cells included normal and H-ras-transformed NIH3T3 fibroblast cells, normal and oncoprotein-overexpressing MCF10A breast cancer cells, and weakly and strongly metastatic cancer cell line pairs originating from human cancers of the skin (A375P and A375SM cells), kidney (SN12C and SN12PM6 cells), prostate (PC3M and PC3MLN4 cells), and bladder (253J and 253JB5 cells). Using magnetic twisting cytometry, cytoskeletal stiffness (g') and internal friction (g″) were measured over a wide frequency range. The dependencies of g' and g″ upon frequency were used to determine the power law exponent x which is a direct measure of cytoskeletal fluidity and quantifies where the cytoskeleton resides along the spectrum of solid-like (x = 1) to fluid-like (x = 2) states. Cytoskeletal fluidity x increased following transformation by H-ras oncogene expression in NIH3T3 cells, overexpression of ErbB2 and 14-3-3-ζ in MCF10A cells, and implantation and growth of PC3M and 253J cells in the prostate and bladder, respectively. Each of these perturbations that had previously been shown to enhance cancer cell motility and invasion are shown here to shift the cytoskeleton towards a more fluid-like state. In contrast, strongly metastatic A375SM and SN12PM6 cells that disseminate by lodging in the microcirculation of peripheral organs had smaller x than did their weakly metastatic cell line pairs A375P and SN12C, respectively. Thus, enhanced hematological dissemination was associated with decreased x and a shift towards a more solid-like cytoskeleton. Taken together, these results are consistent with the notion that adaptations known to enhance metastatic ability in cancer cell lines define a spectrum of fluid-like versus solid-like states, and the position of the cancer cell within this spectrum may be a determinant of cancer progression.

Enhanced analysis of metastatic prostate cancer using stable isotopes and high mass accuracy instrumentation.

The primary goal of proteomics is to gain a better understanding of biological function at the protein expression level. As the field matures, numerous technologies are being developed to aid in the identification, quantification and characterization of protein expression and post-translational modifications on a near-global scale. Stable isotope labeling by amino acids in cell culture is one such technique that has shown broad biological applications. While we have recently shown the application of this technology to a model of metastatic prostate cancer, we now report a substantial improvement in quantitative analysis using a linear ion-trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT) and novel quantification software. This resulted in the quantification of nearly 1400 proteins, a greater than 3-fold increase in comparison to our earlier study. This dramatic increase in proteome coverage can be attributed to (1) use of a double-labeling strategy, (2) greater sensitivity, speed and mass accuracy provided by the LTQ FT mass spectrometer, and (3) more robust quantification software. Finally, by using a concatenated target/decoy protein database for our peptide searches, we now report these data in the context of an estimated false-positive rate of one percent.