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

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 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

Spectroscopic signatures determined using 3D co-culture models can be translated to human breast tissue samples.

<p>(A) Tissue microarray (TMA) biopsy cores (1.5 mm core diameter) were IHC stained for ERα and also imaged using FT-IR imaging (N-H/O-H band at 3300 cm⁻¹ visualized here for clarity). Images classified using Bayesian classifier are displayed as well to highlight the ability of FT-IR to discriminate between cell types in complex samples. Scale bar represents 250 µm. (B) Differences between epithelial pixels in patient samples with high (>80%) and low (<20%) expression of ERα can be seen in peaks at 1080 cm⁻¹ (phosphate) and 1030 cm⁻¹ (glycosidic bonds). Interestingly, there are more apparent differences in these peaks when pixels from fibroblasts are analyzed. Full spectrum (3800 – 950 cm⁻¹), C-H stretching region (3000 – 2750 cm⁻¹), and biochemical fingerprint region (1750 – 950 cm⁻¹) are shown.</p

Treatment of cancerous and normal breast cells with conditioned medium (CM) alters gene expression.

<p>(A) Gene expression changes in MCF-7 cells treated with CM reveal a 10-fold increase in SNAIL, a 7-fold increase in SLUG, and elimination of detectable E-Cadherin mRNA. The increase in EMT markers is accompanied by a down-regulation of ERα mRNA. (B) After treatment of HMEC grown in monolayer culture with CM, there were changes in expression of EMT markers and increases in IL-1β and the growth factor amphiregulin (AREG), suggesting an important role for paracrine signaling between the tumor microenvironment and adjacent normal epithelium.</p

Mammary fibroblasts induce an epithelial-to-mesenchymal transition in ER⁺ breast cancer cells in 3D culture.

<p>(A) Over the course of 6 days in the sandwich co-culture, MCF-7^S display increased mRNA levels of EMT markers SNAIL and SLUG with a decrease in E-cadherin mRNA. (B) Mixed co-cultures were prepared at two fibroblast seeding densities, low- and high-density (relative to MCF-7) and EMT markers were modulated up or down as seen with the sandwich co-culture, and a dose-dependent response to the fibroblast presence was observed. (C) Immunohistochemistry was used to confirm decrease in overall E-cadherin protein level inMCF-7^M (D) Co-culture with human mammary fibroblasts (HMF) increases the invasiveness of MCF-7 breast cancer cells.</p

Fourier transform-infrared (FT-IR) spectroscopic imaging can be used to monitor hormone response in cells in culture.

<p>(A) A change in FT-IR spectroscopic imaging is seen in MCF-7 cells treated with estradiol (E₂), particularly in the C-H stretching region (3000 – 2750 cm⁻¹) and in the peak associated with nucleic acids (1080 cm⁻¹). However, when cells are co-cultured with human mammary fibroblasts (HMF), the response to hormone is lost. (B) Spectral changes are also seen in MCF-7 cells treated with tamoxifen (Tam). While there is a slight induction in peaks associated with the C-H stretching region upon treatment with Tam, the peak associated with nucleic acids, and therefore proliferation, is decreased in MCF-7 cells. This is correlated with the anti-proliferative effects of tamoxifen on ER⁺ cells. In samples that have been co-cultured with HMF, this change is not seen at the 1080 cm⁻¹ peak, corresponding to the endocrine-resistant growth that was seen using proliferation assays.</p

Secreted proteins that were increased 5-fold or higher in co-culture conditioned medium (CM) compared to CM from naïve MCF-7.

<p>Secreted proteins that were increased 5-fold or higher in co-culture conditioned medium (CM) compared to CM from naïve MCF-7.</p