Linking invasive motility to protein expression in single tumor cells

Abstract

The invasion of malignant cells into tissue is a critical step in the progression of cancer. While it is increasingly appreciated that cells within a tumor differ in their invasive potential, it remains nearly unknown how these differences relate to cell-to-cell variations in protein expression. Here, we introduce a microfluidic platform that integrates measurements of invasive motility and protein expression for single cells, which we use to scrutinize human glioblastoma tumor-initiating cells (TICs). Our live-cell imaging microdevice is comprised of polyacrylamide microchannels that exhibit tissue-like stiffness and present chemokine gradients along each channel. Due to intrinsic differences in motility, cell subpopulations separate along the channel axis. The separated cells are then lysed in situ and each single-cell lysate is subjected to western blotting in the surrounding polyacrylamide matrix. We observe correlations between motility and Nestin and EphA2 expression. We identify protein-protein correlations within single TICs, which would be obscured with population-based assays. The integration of motility traits with single-cell protein analysis - on the same cell - offers a new means to identify druggable targets of invasive capacity.

Two high-yield complementary methods to sort cell populations by their 2D or 3D migration speed

“…When they exist, they are either of very low throughput, or difficult to implement or parallelize. One such method was developed by Lin et al ., where polyacrylamide microfluidic channels were employed to segregate cells based on their motility under a chemokine gradient, followed by protein expression analysis by in situ Western blotting ( Lin et al , 2018 ). In another work, the ability of migrating cells to phagocytose fluorescent particles from a substrate was used to study and sort cells using fluorescence-activated cell sorting (FACS; Windler-Hart et al , 2005 ).…”

A novel xenograft model reveals invasive mesenchymal transition and ineffective angiogenic response during anti-angiogenic therapy resistance

“…We then performed further investigation of the invasive potential of U87-Bev R and U87-Bev S cells in threedimensional bioengineered models. First, we studied these cells in hydrogel (16) platforms that modeled the gray and white matter along which peritumoral invasion occurs and contained the hyaluronic acid shown to be upregulated in the extracellular matrix of bevacizumab-resistant tumors (17). In these hydrogel platforms, U87-BevR cells proved more invasive than U87-BevS cells (P<0.001; Fig.…”

“…The microchannel device was fabricated as described by Lin et al (16). Briefly, the micropatterned polyacrylamide (PA) gel (8%T, 3.3%C acrylamide/bisacrylamide (Sigma-Aldrich) was activated with Sulfo-SANPAH (Thermo Scientific) and incubated with a 1:2 dilution of Matrigel (Fisher Scientific, Cat# CB40230, diluted in 10% FBS cell culture medium) for 24 hours at 4°C (see Supplementary Methods).…”

Microfluidic Analyzer Enabling Quantitative Measurements of Specific Intracellular Proteins at the Single-Cell Level

“…More specifically, barcoding microchips with a commercial brand of “Isoplexis” were developed where individual cells are confined within microchambers, and the absolute quantification of specific intracellular proteins is realized by cell lysis and the captures of target cellular proteins by preprinted antibodies on the bottom surfaces of the microchips [ 14 , 15 , 16 , 17 ]. In a second microfluidic approach, with a commercial brand of “single-cell westerns”, key steps of settling single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing are included, enabling the quantitative analysis of cellular proteins [ 18 , 19 , 20 ]. However, in comparison to flow cytometry, these microfluidic instruments still suffer from limited throughputs since they cannot characterize single cells in a continuous fluid flow.…”