Abstract
Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image cultured cells and membrane-bound microbeads in twelve independently-focusing channels simultaneously, visiting all wells in eight steps. We use the AHTM and passive bead rheology techniques to determine the relative compliance of human pancreatic ductal epithelial (HPDE) cells, h-TERT transformed HPDE cells (HPNE), and four gain-of-function constructs related to EMT. The AHTM found HPNE, H-ras, Myr-AKT, and Bcl2 transfected cells more compliant relative to controls, consistent with parallel tests using atomic force microscopy and invasion assays, proving the AHTM capable of screening for changes in mechanical phenotype.
Highlights
Steady increases in the availability and use of high-throughput (HT) systems has accelerated progress in pursuits as diverse as mapping the human genome, studying cancer chemotherapeutic drug-resistance at the individual cellular proteomics level, and identifying pathogenic bacteria in the hospital setting[1,2,3]
We describe testing the utility of the array high-throughput microscope (AHTM) in identifying subtle changes in cell compliance produced by the expression of gain-of-function (GOF) versions of single genes involved in cancer biology
We found that the AHTM could consistently distinguish both the relatively subtle changes engendered by single genetic changes, and the larger difference between normal and oncogene-expressing cells
Summary
Steady increases in the availability and use of high-throughput (HT) systems has accelerated progress in pursuits as diverse as mapping the human genome, studying cancer chemotherapeutic drug-resistance at the individual cellular proteomics level, and identifying pathogenic bacteria in the hospital setting[1,2,3]. Increased compliance, have been correlated with increased invasiveness in many systems, primarily by atomic force microscopy (AFM), magnetic twisting cytometry and magnetic tweezers[6,8,9,10,11,12,13,14] These techniques, while providing excellent measures of compliance, are low-throughput and time-intensive. We introduce an array high-throughput microscope (AHTM) that can assess the stiffness of cancer cells grown in monolayer culture It combines twelve independently-focusing objectives, two-color epi-fluorescence, high frame rate imaging, automated analysis and lossless data compression. The GOF was functionally validated in the stable cell lines using standard methods, the results of which are shown as Supplementary Figs 1 and 2 We compared those changes with an h-TERT immortalized, nestin and K-Ras expressing pancreatic ductal epithelial line (HPNE)[34]. We found that the AHTM could consistently distinguish both the relatively subtle changes engendered by single genetic changes, and the larger difference between normal and oncogene-expressing cells
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