Nanowire substrate-based laser scanning cytometry for quantitation of circulating tumor cells.

We report on the development of a nanowire substrate-enabled laser scanning imaging cytometry for rare cell analysis in order to achieve quantitative, automated, and functional evaluation of circulating tumor cells. Immuno-functionalized nanowire arrays have been demonstrated as a superior material to capture rare cells from heterogeneous cell populations. The laser scanning cytometry method enables large-area, automated quantitation of captured cells and rapid evaluation of functional cellular parameters (e.g., size, shape, and signaling protein) at the single-cell level. This integrated platform was first tested for capture and quantitation of human lung carcinoma cells from a mixture of tumor cells and leukocytes. We further applied it to the analysis of rare tumor cells spiked in fresh human whole blood (several cells per mL) that emulate metastatic cancer patient blood and demonstrated the potential of this technology for analyzing circulating tumor cells in the clinical settings. Using a high-content image analysis algorithm, cellular morphometric parameters and fluorescence intensities can be rapidly quantitated in an automated, unbiased, and standardized manner. Together, this approach enables informative characterization of captured cells in situ and potentially allows for subclassification of circulating tumor cells, a key step toward the identification of true metastasis-initiating cells. Thus, this nanoenabled platform holds great potential for studying the biology of rare tumor cells and for differential diagnosis of cancer progression and metastasis.

Cell adhesion and migration on nanopatterned substrates and their effects on cell-capture yield.

With scanning electron microscopy analysis, we investigated the role of nanoscale topography on cellular activities; e.g. cell adhesion and spreading by culturing A549 cells (human lung carcinoma cell line cells) for 1-48 h on three sets of nanostructures; quartz nanopillars (QNPs), silicon nanopillars and silicon nanowire (SiNW) arrays, along with planar glass substrates. We found that cells on QNP arrays developed a longer shape than those on SiNW arrays. In addition, we studied how cell morphologies influence the cell-capture yield on the three sets of nanostructures. This research showed that the filopodial formations were directing the cell-capture yield on nanostructured substrates. This finding implies the possibility of using nanoscale topography features to control the filopodial formation including extension and migration from the cells. Using streptavidin-functionalized SiNW substrate, we further demonstrated a substantially higher yield (~91.8 ± 5.9%) than the planar glass wafers (~24.1 ± 7.5%) in the range of 200-3000 cells.

Filopodial morphology correlates to the capture efficiency of primary T-cells on nanohole arrays.

Nanostructured surfaces emerge as a new class of material for capture and separation of cell populations including primary immune cells and disseminating rare tumor cells, but the underlying mechanism remains elusive. Although it has been speculated that nanoscale topological structures on cell surface are involved in the cell capture process, there are no studies that systematically analyze the relation between cell surface structures and the capture efficiency. Here we report on the first mechanistic study by quantifying the morphological parameters of cell surface nanoprotrusions, including filopodia, lamellipodia, and microvilli in the early stage of cell capture (< 20 min) in correlation to the efficiency of separating primary T lymphocytes. This was conducted by using a set of nanohole arrays (NHAs) with varying hole and pitch sizes. Our results showed that the formation of filopodia (e.g., width of filopodia and the average number of the filopodial filaments per cell) depends on the feature size of the nanostructures and the cell separation efficiency is strongly correlated to the number of filopodial fibers, suggesting a possible role of early stage mechanosensing and cell spreading in determining the efficiency of cell capture. In contrast, the length of filopodial filaments was less significantly correlated to the cell capture efficiency and the nanostructure dimensions of the NHAs. This is the first mechanistic study on nanostructure-based immune cell capture and provides new insights to not only the biology of cell-nanomaterial interaction but also the design of new rare cell capture technologies with improved efficiency and specificity.

A quartz nanopillar hemocytometer for high-yield separation and counting of CD4(+) T lymphocytes.

We report the development of a novel quartz nanopillar (QNP) array cell separation system capable of selectively capturing and isolating a single cell population including primary CD4(+) T lymphocytes from the whole pool of splenocytes. Integrated with a photolithographically patterned hemocytometer structure, the streptavidin (STR)-functionalized-QNP (STR-QNP) arrays allow for direct quantitation of captured cells using high content imaging. This technology exhibits an excellent separation yield (efficiency) of ~95.3 ± 1.1% for the CD4(+) T lymphocytes from the mouse splenocyte suspensions and good linear response for quantitating captured CD4(+) T-lymphoblasts, which is comparable to flow cytometry and outperforms any non-nanostructured surface capture techniques, i.e. cell panning. This nanopillar hemocytometer represents a simple, yet efficient cell capture and counting technology and may find immediate applications for diagnosis and immune monitoring in the point-of-care setting.