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.

Figure 1

SCAMPR assay correlates cell migratory phenotype and protein expression. Microfluidic integration supports a Single-Cell Analysis of Motility and Proteotype (SCAMPR) assay. A heterogeneous population of primary cells is first dissociated into a single-cell suspension. The cells are then seeded into the SCAMPR device and tracked as each cell chemotactically migrates through the channels under a chemokine gradient, which reports motility and motility-related parameters, persistence and average aspect ratio. Immediately following the live-cell tracking, cells are immobilized in an agarose layer and the scWB is run in order to measure protein expression on each tracked cells. Motility and proteotype information from single cells is then correlated to associate proteomic markers with invasive motility properties.

Figure 2

SDF-1 chemokine gradient resolves two cell lines with known differences in motility. a) Time-averaged FITC-Dextran fluorescence signal in the SCAMPR device. Linear regression of average gradient shows a slope of −0.00166 RFU per μm and a R² value of 0.996. The average gradient curve is calculated from 33 gradients from 3 separate devices. Error bars represent standard deviation calculated from 33 gradients from 3 separate devices. RFU: relative fluorescence unit. b) Migration-induced separation of a mixed population of labeled Control and DN Rac1 cell lines. Empty Vector: 43 cells from 3 independent experiments; DN Rac1: 23 cells from 3 independent experiments.

Figure 3

Agarose cell-encapsulation reduces dilution of single-cell lysates. a) Representative images of fluorescently labeled GBM TICs during in-microchannel lysis with (Agarose) and without (Open) an agarose encapsulating layer. TICs were labeled with CMFDA Cell Tracker dye. Lines indicate the edges of the microchannel. Scale bar = 20 μm. b) Time course of the total integrated fluorescence signal from labeled GBM TICs lysed in the Open and with Agarose conditions. At the start of electrophoresis (indicated by the red line), the agarose lid improved lysate retention by 72% relative to the open condition. Error bars represent standard deviation calculated from 3 independent experiments. c) Representative fluorescence trace (black) of a cell at the beginning of lysis (0 s) and the corresponding Gaussian fit line (blue) with the σ value shown. The dashed line in the insert denotes the axis of the fluorescent trace. d) Time course of the normalized peak width quantified from the fitted Gaussian curve of the fluorescence intensity. At the start of the electrophoresis at 15 s, the agarose condition shows an 84% decrease in peak width compared to the open condition. Error bars represent standard deviation calculated from 3 independent experiments.

Figure 4

Persistence, but not aspect ratio, is correlated with TIC speed in the SCAMPR device. a) Dot plot showing the individual TIC speeds across 9 devices for a GBM TIC line. Each point represents a single cell, with a global maximum TIC speed of ~170 μm per hr and a global minimum of 7.4 μm per hr. Lines represent the mean TIC speed per device. Scatter plots reveal that the TIC persistence (b), but not average aspect ratio (c) is significantly correlated with TIC speed (Spearman’s rank r_Persistence=0.676, p<0.0001, n=68 cells; r_{Aspect Ratio}=−0.134, p=0.430, n=37 cells).

Figure 5

SCAMPR assay reveals novel protein expression correlations in GBM TICs. a) Representative fluorescence micrographs and intensity plots from the SCAMPR assay of GBM TICs. Arrows indicate peak location. Scale bars represent 100 μm. b) Biaxial scatter plots report protein expression for all markers for each GBM TICs from low, medium and high motility TIC subpopulations. Motility subpopulations were created based on three equally sized intervals of speed ranging from 0 μm per hr to 170 μm per hr (maximum observed TIC speed). TICs were binned into motility subpopulations based on magnitude of speed (Low Motility: 0–56.6 μm per hr, Medium Motility: 56.6–113.3 μm per hr, High Motility: 113.3–170 μm per hr). Blue squares, black triangles and red circles represent low, medium and high motility subpopulations, respectively.

Figure 6

SCAMPR assay reveals Nestin and EphA2 correlate with TIC speed. Scatter plots of individual TICs show that (a) Nestin and (b) EphA2 are positively correlated with TIC speed (Spearman’s rank r_Nestin=0.381, p=0.001, n=68 cells; r_EphA2=0.451, p=0.040, n=21 cells). However, (c) STAT3 and (d) β-tubulin are not correlated with TIC speed (Spearman’s rank r_STAT3=0.030, p=0.848, n=43 cells; r_β-tubulin=−0.002, p=0.990, n=34 cells).

Figure 7

SCAMPR assay allows for joint multivariable analysis of phenotype and proteotype. a) Relative expression levels of Nestin, EphA2, STAT3, and β-tubulin in single GBM TICs with respect to cell speed. Only the subset of TICs with quantifiable expression levels for all four proteins is displayed in this plot. Protein expression in each TIC is normalized to the strongest signal for each protein. Within each row, tile color corresponds to relative expression level with black being the lowest and white being the highest. Each column represents the proteotype for one TIC and the TICs are organized in increasing cell speed order. b) Representative cell trajectories of single TICs expressing either low or high Nestin or EphA2. High and Low expression levels represent the top 50% and bottom 50% expression level for each respective protein.