Quantifying heterogeneity and dynamics of clonal fitness in response to perturbation

Abstract

The dynamics of heterogeneous clonal lineages within a cell population, in aggregate, shape both normal and pathological biological processes. Studies of clonality typically relate the fitness of clones to their relative abundance, thus requiring long-term experiments and limiting conclusions about the heterogeneity of clonal fitness in response to perturbation. We present, for the first time, a method that enables a dynamic, global picture of clonal fitness within a mammalian cell population. This novel assay allows facile comparison of the structure of clonal fitness in a cell population across many perturbations. By utilizing high-throughput imaging, our methodology provides ample statistical power to define clonal fitness dynamically and to visualize the structure of perturbation-induced clonal fitness within a cell population. We envision that this technique will be a powerful tool to investigate heterogeneity in biological processes involving cell proliferation, including development and drug response.

Figure 1

Schematic of clonal Fractional Proliferation experimental workflow. (a) Cells are seeded at single-cell density into microtiter imaging plates. Single cells are allowed to proliferate for 6 days in full growth media to expand into colonies. Once colonies have reached an optimal size, cells are imaged and then the experimental perturbation is immediately added. Subsequently, each well is imaged daily until the end of the experiment. (b) Spatially- and temporally registered images facilitate tracking of individual colonies. Time-ordered stacks of image montages allow sequential measurements of colony cell numbers during drug treatment. (c) Fluorescent images of cell nuclei for single-cell derived PC9 colonies. Sequential images show that single-cells form colonies when cultured in full growth media (Untreated). Tracking colonies after addition of cycloheximide (Chx, 500 ng/ml) or DMSO (control), individual colonies shows drug-induced changes in cell number over time.

Figure 2

Validation of cFP image processing. (a) Top row: Images of a representative colony throughout cycloheximide (Chx, 500 ng/ml) treatment at the indicated days. Middle row: Binary mask generated in ImageJ using the same intensity threshold at all time points. Bottom row: Identified cells after image processing. (b) Automated counting of cell nuclei appropriately quantifies colony cell number. Manual cell counts from colony images at various time points (n = 219) are used as a reference to validate the automatically measured colony cell numbers. The superimposed line represents the linear model fit for the data, with the corresponding adjusted R-squared value (adj. R²) (c) Q-Q plot of the residuals of the linear model fit in used in (b). There is insufficient bias to conclude that automated image analysis is inappropriate for colonies of certain size.

Figure 3

Perturbation-induced proliferation (PIP) rates capture clonal fitness. (a) Cycloheximide (Chx, 500 ng/ml) reduces PC9 proliferation relative to control. Each data point represents the average number of cycloheximide-treated cells relative to control (n = 4 wells from a cFP experiment). (b) Histogram of the per-colony relative log₂ fold change in cell number after 10 days of cycloheximide treatment. Curve represents the best fit using a skew-normal distribution. (c) Cycloheximide-induced colony dynamics. Colony doublings represents the log₂-transformed cell number per colony normalized to the initial size of that colony. Boxplots show the interquartile range of cycloheximide-treated colony doublings at each time point. Colored lines display the dynamics of three representative colonies in d. (d) Images of H2B-mRFP labeled cell nuclei for the representative colonies in c. Scale bars = 100 µm (e) Colony dynamics, same as in c, except colonies are normalized to the colony size after 3 days of treatment. Lines and boxplots are the same as in c. (f) Linear model fits of colony dynamics simplify the colony doublings data. The linear model fit of each colony response from 3 days on is shown as a gray line. The estimated linear model fits of the representative colonies from c are shown as colored lines, as previously described. Boxplots represent the interquartile range of the expected values for colony doublings at each time point.

Figure 4

Measuring the clonal structure in response to perturbations. (a) Concentration dependent effects of cycloheximide treatment. Histograms of PIP rates are shown for PC9 colonies at decreasing concentrations (left to right). Curves represent the estimated skew-normal probability distribution with the indicated values. Kolmogorov-Smirnov test p-values are shown. High p-value indicates there is insufficient evidence to reject the fit (b). Upper-left, The skew-normal fits (same color) from a are superimposed to illustrate the concentration-dependent effects of cycloheximide without the data. Concentration dependent effects are similarly shown for trametinib (Trm), SB203580 (SB), and anisomycin (An). (c) Skew normal fits are displayed for A375 cells treated with either ABT-737 (ABT) or PLX4720 (PLX).

Figure 5

Investigating PIP rates using discrete sublines. (a) PC9 colonies with unique proliferation rates cultured in DMSO imaged at the indicated time points. All cells are fluorescently labeled with both Histone H2B-RFP (red pseudocolor) and geminin-mAg (green pseudocolor). Green cell nuclei indicate cells that have passed the G1/S transition; red cell nuclei mark cells that have not. Scale bars = 100 microns. (b) Distribution PC9 of DMSO-treated PIP rates. Arrows indicate the PIP rate of the color-matched colonies from a. (c) Quantitation of proliferation rates for two representative PC9 discrete sublines, DS3 and DS5 (values are the average of triplicate wells from n = 3 experiments). Error bars = SE. (d) Representative images from the experiments in c. (e) Percent of cells in S/G2/M phase (geminin-mAg) during DMSO treatment (data are the average of triplicate wells in n=3 experiments). Error bars = SE. (f) Quantitation of single-cell speed for DS3 and DS5 treated with DMSO. (g) Quantitation of single-cell EGFR expression from immunofluorescence images using CellAnimation. Curves represent the best fit using a skew normal distribution. * p < 0.05; ** p < 0.01; *** p < 0.001.