Highly Sensitive Quantitative Imaging for Monitoring Single Cancer Cell Growth Kinetics and Drug Response

<div><p>The detection and treatment of cancer has advanced significantly in the past several decades, with important improvements in our understanding of the fundamental molecular and genetic basis of the disease. Despite these advancements, drug-screening methodologies have remained essentially unchanged since the introduction of the <i>in vitro</i> human cell line screen in 1990. Although the existing methods provide information on the overall effects of compounds on cell viability, they are restricted by bulk measurements, large sample sizes, and lack capability to measure proliferation kinetics at the individual cell level. To truly understand the nature of cancer cell proliferation and to develop personalized adjuvant therapies, there is a need for new methodologies that provide quantitative information to monitor the effect of drugs on cell growth as well as morphological and phenotypic changes at the single cell level. Here we show that a quantitative phase imaging modality known as spatial light interference microscopy (SLIM) addresses these needs and provides additional advantages over existing proliferation assays. We demonstrate these capabilities through measurements on the effects of the hormone estradiol and the antiestrogen ICI182,780 (Faslodex) on the growth of MCF-7 breast cancer cells. Along with providing information on changes in the overall growth, SLIM provides additional biologically relevant information. For example, we find that exposure to estradiol results in rapidly growing cells with lower dry mass than the control population. Subsequently blocking the estrogen receptor with ICI results in slower growing cells, with lower dry masses than the control. This ability to measure changes in growth kinetics in response to environmental conditions provides new insight on growth regulation mechanisms. Our results establish the capabilities of SLIM as an advanced drug screening technology that provides information on changes in proliferation kinetics at the cellular level with greater sensitivity than any existing method.</p></div

Measurement of cancer cell proliferation using SLIM dry mass measurements.

<p>(A) Schematic of experimental setup. A fully automated commercial phase contrast microscope equipped with stage top incubation control and x, y, z-scanning capabilities was used to scan a 1.5 mm×1.2 mm area in each well of a 2-well slide every 30 minutes. The components in the dotted line comprise the SLIM add-on module: Fourier Lens 1 (FL1) projects the pupil plane of the phase contrast microscope onto a Liquid Crystal Phase Modulator (LCPM), which provides control over the phase delay between the scattered and un-scattered light; Fourier Lens 2 projects the phase-modulated image onto a CCD. All components of the instrument were synchronized using the CPU. (B) Representative images of a scanned field of view in one of the chambers at 0 hours and 94 hours, the area in the dashed yellow line is enlarged and shown at each time point (yellow scale bar is 50 microns). (C) Average normalized surface area for clusters in each group in the labelled time periods. (D) Average normalized mass for clusters in each group in the labelled time periods. (C–D) Square markers indicate mean, centerline is median, top of box is 25^th percentile line, bottom is 75^th percentile line, whiskers indicate 5^th and 95^th percentiles, significance was tested using an un-paired t-test, o: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001. (E) WST-1 proliferation assay measurement at 72 hours.</p

Growth data for all clusters.

<p>(A) Normalized mass vs. Time for all clusters that were analyzed. (B) Normalized area vs. time for all clusters. (A–B) Dotted lines show individual cluster data and solid lines show averaged data. Dashed lines indicate where the difference between groups becomes significant.</p

Cluster growth rate analysis.

<p>(A) Dry mass density maps of representative clusters from each group of MCF-7 breast cancer cells at every 22 hours. The colors indicate the dry mass density at each pixel as shown on the color bar. The yellow scale bar is 50 microns. Note that in the E2 + ICI group, ICI was added to each sample at 10 hours. (B) Cluster growth rate in each group in the shown time period. (C) Cluster growth rate in each group as a function of normalized mass. Solid lines are shown as a guide to the eye to determine how the growth rate is changing as a function of mass growth. (B–C) Square markers indicate mean, centerline is median, top of box is 25^th percentile line, bottom is 75^th percentile line, whiskers indicate 5^th and 95^th percentiles, significance was tested using an un-paired t-test, o: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001.</p

Estrogen modulated changes in proliferation kinetics.

<p>(A) Doubling time in each group, the mean doubling time is reduced by 12 hours in the E2 group compared to the Veh and E2 + ICI groups, indicating that adding ICI returns the doubling time to control levels. (B) Percent change in the mean cell mass over the measurement period for each group. A significant decrease in the cell mass is observed in both the E2 and E2+ICI groups compared to the control. (C) Measured doubling time vs. change in mean cell mass for each cluster that was measured, these two parameters can be used to separate the three groups completely and can serve as a growth signature.</p

Measurement of prominent structures found in 26 cells.

<p>In the x–y plane a coil structure is visible that has a period of approximately 0.43 µm and does not vary with the length of the cell. In the x-z plane another structure is visible that has a period of half the cell-length.</p

Sparsity property of phase images.

<p>Images show the original phase image, and the output images obtained by applying first order directional derivatives in the x, y, and z directions, as labeled, scale bar is 1 µm. The plot shows the corresponding log-histograms, the increase in sparsity is clearly visible.</p

Three dimensional point spread function.

<p>A) Comparison of raw and deconvolved. PSF in the x-y plane; the deconvolution process reduces the FWHM from 397 nm to 153 nm. B) Comparison of raw and deconvolved PSF in the x–z plane; the deconvolution process reduces the FWHM from 1218 nm to 357 nm. The dashed lines show the data and the circular markers indicate the Gaussian fit used to determine the FWHM.</p

Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots

Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on–off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (<i>i.e.</i>, SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on-state, ∼4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor <i>in vitro</i>, and the improved labeling efficiency enables tracking of single kinesins in live cells