High Content Imaging for Genotoxicity

Microscopy is the preferred experimental tool to study cellular mechanisms of DNA-damage induction or genotoxicity. This is because primary outcomes of genotoxicity, e.g. DNA double-strand breaks or micronuclei, can be readily detected via imaging. For DNA double-strand break detection, antibodies exist that can stain DNA-repair molecules that accumulate around physical DNA damage and form detectable spots called DNA-damage foci. Micronuclei are extranuclear DNA compartments that can be detected with standard DNA stains like DAPI.

The cell cycle (simple overview shown in Fig. 1A) also plays a profound role in understanding mechanisms of DNA-damage. First, cells are responding to DNA-damage via cell cycle arrest, hence shifts in cell cycle phases are indicative of a drug’s efficacy to inflict DNA-damage. Also, certain cell cycle phases are more susceptible to DNA-damage. The genome is especially vulnerable to genotoxic conditions mainly during DNA synthesis (S-phase) and during mitosis (M-phase) because DNA is actively being processed (by replication or segregation). Third, some drugs act exclusively on specific cell cycle phases, because they target cell-cycle specific molecular mechanisms (e.g. in replication).

Taken together, the experimental settings to perform DNA-damage analysis are quite complex and require careful execution of imaging and image analysis.

To validate the correct separation of nuclei into cell cycle phases, we plotted EdU total nuclear intensity versus DAPI total nuclear intensity to obtain the characteristic cell cycle distribution of dividing cells (Figure 2B).

Cytotoxic Efficacy

The first and most important read-out for drug testing in oncology is determining the dose-response curve. Low concentrations of a drug typically are tolerated and do not affect the cell proliferation, while doses beyond a certain threshold will kill most cells in a culture. This leads to a dose-response curve with a characteristic sigmoidal shape. The center of this distribution marks the point, where the population is reduced to 50%. This point is typically called “Inhibitory Concentration 50” (IC50) and is a characteristic measure for a drug to describe its cytotoxic efficacy.

In this dataset, determining the dose-response curve can be done simply by determining total numbers of nuclei per sample. This can be obtained in ZEISS arivis Pro using the Object Manager and by creating group statistics. To obtain a suitable curve fitting, cell numbers were extracted and analyzed with a statistical software (Figure 3A). In this data set, the IC50 was 146.5 nM, which is a quite potent cytotoxicity. Note also, that the change in survival occurs quite abruptly. Cells are largely unaffected by the drug at 75 nM, but die at 250 nM. These concentrations will serve as reference points for the following discussions.

Cell-cycle Dependent DNA-damage Elucidates Mode of Cell Killing

Cell proliferation has a major influence in the efficacy of genotoxic drugs, as DNA is more vulnerable in the DNA synthesis phase (S-phase) or during mitosis. To approach a deeper mechanistic understanding of the genotoxic effects in this data set, it is worth taking cell-cycle phases into account. The first plot simply shows the cell cycle distributions in each sample (Figure 5A). High doses (>250 nM) cause the fraction of actively cycling cells (S / G2 / M phase) to increase from 15-20% to 40-50%. This is at first glance puzzling, because other types of genotoxic treatments (e.g. ionizing irradiation) instead cause proliferative arrest which leads to higher fractions of G0/G1 cells.

To evaluate this further, we also checked DNA-damage formation in different cell-cycle phases (Figure 5B-D). High focus numbers occur specifically at high drug doses in S-phase and G2/M-phase. Hence, high amounts of DNA-damage impair these cells in S- and G2/M-phase and prevent them from completing the cell-cycle, with a larger fraction of cells is in these phases. In fact, this was confirmed by another experiment not included here (by measuring replication speed). In summary, we observe very clear indications that the genotoxic drug is in fact a drug specifically acting on dividing cells, with the major driving force for cytotoxicity being the overwhelming accumulation of DNA-damage in these phases.

Both intense DNA-damage foci and micronuclei only form after (S-phase damage) cells passage through mitosis. This explains why this phenomenon is restricted to intermediate doses. For higher doses, cells completely fail to complete mitosis, hence no micronuclei and high-intensity DNA-damage foci are able to form.

Large DNA-damage foci, as opposed to small foci, form at genomic sites with complex DNA-damage (e.g. consisting of several double-strand breaks or involving multiple chromosomes) that cannot be repaired easily. Such damage is to be expected during micronucleus formation. This provokes the question of whether large DNA-damage foci and micronuclei are two consequences of the same process. To analyze this, the correlation of micronuclei and large DNA-damage foci was determined at a single cell level. Cells were first separated into MN-positive and MN-negative groups; the average of the most intense DNA-damage foci per cell was plotted (Figure 6F). Then, cells were stratified according to their most intense DNA-damage focus and the MN-positive cell fraction was plotted (Figure 6G). Both plots show that cells with high DNA-damage focus intensities (> 200000) also show the highest fraction of MN-positive cells. An example of how both phenomena co-occur is shown in Figure 6H.