Docetaxel treatment induces cell death both in vitro and in vivo

For our in vitro and in vivo studies, we chose to use two colorectal tumor cell lines that can be studied in vitro and grow tumors upon injection in mice. We used the C26 and SW480 cell lines as a mouse isograft and human xenograft colorectal tumor model respectively. To confirm that both cell lines are sensitive to treatment with the semi-synthetic taxane docetaxel in vitro, we determined cell viability after several days of treatment with increasing doses of docetaxel. Colony formation capacity was clearly affected in both cell lines at a dose of 2–3 nM docetaxel, indeed showing the potency of this chemotherapeutic to kill tumor cells in tissue culture (Figure 1A).

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Figure 1. Docetaxel kills tumor cells in vitro and in vivo.

A, colony formation assay of indicated cell lines treated with increasing doses of docetaxel (DTX). Colony formation capacity was determined relative to untreated controls 6 days after a single addition of docetaxel. B, quantification of the number of apoptotic cells before or 2–4 days after a single intravenous injection of 25 mg/kg docetaxel. Average of 3 independent experiments per cell line + SEM is indicated. Vehicle (PBS)-treatment is shown in Figure S1C.

https://doi.org/10.1371/journal.pone.0064029.g001

In order to confirm the docetaxel effect in vivo, we subcutaneously injected C26 and SW480 cells in BalbC and immune-compromised SCID mice respectively and allowed the cells to form a tumor within 2–6 weeks. Once tumors were detectable by palpation, we treated animals once with the maximum tolerated dose of docetaxel (25 mg/kg) (Figure S1A) and visualized the number of apoptotic cells (defragmented cells) by intravital imaging (Figure S1B). In line with our in vitro data, we observed a substantial increase in the percentage of apoptotic cells in both tumor models (Figure 1B), which was not present in vehicle (PBS)-treated tumors (Figure S1B, C). From these data we conclude that our tumors models are sensitive to docetaxel both in vitro and in vivo.

Simultaneously imaging mitotic progression and the onset of apoptosis

To unequivocally prove that docetaxel-induced apoptosis is mediated through defects in mitotic progression, both processes should be visualized simultaneously in the same cells in vitro and in vivo before and after docetaxel treatment. Here, we have developed an imaging method to achieve this (Figure 2A and B).

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Figure 2. Simultaneous imaging of mitosis and the induction of apoptosis.

A, schematic representation of the caspase-3 FRET probe that reports the early induction of apoptosis. In the absence of caspase-3 activity, excited CFP molecules will transfer their energy to YFP, resulting in short CFP fluorescence lifetime and YFP emission. A rise in caspase-3 activity, indicative of apoptosis induction, results in the cleavage of the DEVD motif in between the CFP and YFP moieties. This results in loss of FRET, an increase in CFP-YFP ratio and an increased CFP fluorescence lifetime. B, schematic representation of photo-marking of H2B-Dendra2 (H2B-D) cells. Switching of H2B-D from green to red enables the tracking of single cells and visualization of mitotic progression. C, color scheme and representative cells (alive, early apoptotic, late apoptotic) in vitro and in vivo showing CFP-YFP ratio changes. D, in vitro cells representative of H2B-D switching and mitotic progression. E, left: Representative image of H2B-D photo-switching of SW480 tumor cells in vivo. Scale bar represents 50 µm. Right: Stills of individual SW480 cell in vivo tracked at consecutive days.

https://doi.org/10.1371/journal.pone.0064029.g002

Activation of the protease caspase-3 is crucial for apoptosis induction and catalyzes the cleavage of several key cellular proteins. Caspase-3 activation precedes chromosome condensation and cell fragmentation, the typical morphological changes associated with apoptotic cell death [22]. We and others have recently used a caspase-3 Fluorescent Resonance Energy Transfer (FRET) probe to visualize the onset of apoptosis in vitro and in vivo [20], [23]–[25]. The caspase-3 FRET sensor consists of a CFP and YFP moiety separated by the caspase-3 DEVD cleavage motif and is targeted to the plasma membrane by a C-terminal CAAX sequence [26] (Figure 2A and C). Under normal conditions, caspase-3 is inactive and CFP and YFP are in close proximity, so that CFP can transfer its energy to YFP. As a result of this energy transfer, CFP fluorescence will drop and YFP fluorescence will increase, leading to a low CFP to YFP ratio (referred to as CFP-YFP ratio). Caspase-3-dependent cleavage of the DEVD motif will perturb energy transfer from CFP to YFP and result in an increased CFP-YFP ratio. We created both C26 and SW480 cell lines stably expressing this apoptosis sensor and, as expected, we were able to visualize apoptosis induction both in vitro and in vivo using this caspase-3 FRET sensor (Figure 2C).

To follow both caspase-3 activation and mitotic progression before and after docetaxel treatment, we, in addition to the caspase-3 biosensor, also stably introduced Histone 2B tagged to photo-switchable Dendra2 [27] (H2B-D) in C26 and SW480 cell lines (Figure 2B and D). Mitosis can be identified when chromosomes and therefore H2B-Dendra condense. Cells without condensed H2B-Dendra were scored as interphase. In prior studies, we have used Dendra2 to specifically photo-mark and trace individual cells [28]–[30]. By switching the color of H2B-Dendra2 (H2B-D) from green to red using a violet laser [27] (Figure 2B and D), we were able to mark cells and track them both in vitro and in vivo over multiple imaging sessions (Figure 2D and E). Photo-switching of Dendra2 did not affect caspase-3 FRET measurements (Figure S2A) and more importantly, FRET levels did not change during mitotic progression, excluding possible effects of the mitotic state on FRET efficiency (Figure S2B).

To determine the apoptotic CFP-YFP ratio, the ratio at which caspase-3 activity and early apoptosis are induced, we determined the relative CFP-YFP ratio (see Materials and Methods) in cells positively and negatively stained for cleaved caspase-3 (activated form). On average, the CFP-YFP ratio was 1.3 times higher in the cleaved caspase-3-postive when compared to negative cells (Figure S2C). From here on, cells with a relative CFP-YFP ratio higher than 1.3 will be referred to as cells with an apoptotic CFP-YFP ratio.

These data show that by imaging the caspase-3 FRET sensor and H2B-Dendra2 simultaneously, we are able to visualize mitotic progression and the onset of apoptosis (1.3 times increased CFP-YFP ratio) in the same cells both in vitro and in vivo.

Docetaxel treatment induces caspase-3 activation both in vitro and in vivo

Next, we determined the onset of apoptosis in our cell lines after docetaxel administration in vitro. The CFP-YFP ratio clearly increased in mitotic cells after prolonged docetaxel treatment (Figure 3A). Importantly, cells that were morphologically apoptotic (disintegrated) showed an even higher CFP-YFP ratio (5.6) when compared to the rest of the mitotic cells treated with docetaxel (3.7). This indicates that we can observe the docetaxel-induced increase in caspase-3 activity well before cells undergo the typical morphological changes associated with apoptosis (Figure 2C), which is in line with a recently proposed model in which caspase-3 activity slowly increases during a mitotic delay imposed by treatments with anti-mitotic drugs [10].

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Figure 3. Docetaxel increases caspase-3 activity in vitro and in vivo.

A, quantification of the CFP-YFP ratio of SW480 cells in vitro under indicated conditions (1 µm docetaxel). The 24* indicates that only apoptotic cells were analyzed, in all other cases (0, 16, and 24) mitotic cells were quantified. Apoptotic and mitotic cells were determined according to morphology. Apoptotic: membrane blebbing, fragmented DNA. Mitotic: condensed DNA, rounded membrane morphology. 10 cells were quantified per condition. Average + SEM is shown. B, quantification of the CFP-YFP ratio upon intravital imaging of tumor cells stably expressing caspase-3 FRET sensor. The CFP-YFP ratios (normalized to average CFP-YFP value before treatment) of individual cells in vivo of the same photoswitched tumor fields were plotted against the indicated times after a single intravenous injection of 25 mg/kg docetaxel. Red dotted line represents the average normalized CFP-YFP ratio of vehicle (PBS)-treated mice as shown in Figure S3A. Results of 3 independent experiments (visualized by different symbols for different mice) per cell line are shown. One symbol represents one cell. Line indicates median + IQR. ***: significant (Mann Whitney U test, p<0.008). C, left: Color scheme of CFP fluorescence lifetime analysis as explained in Figure 2A and C. Short lifetimes (blue) indicate high FRET levels, long lifetimes (red) indicate low FRET levels. Right: Representative FLIM images of a C26 tumor showing a group of tumor cells before (−) (upper panel) and 20 hours after (+) (lower panel) 25 mg/kg docetaxel treatment. D, the fluorescent lifetime of C26 cells in vivo plotted at indicated time points after docetaxel treatment. The red dotted line represents the average lifetime of vehicle (PBS)-treated mice as shown in Figure S3B. One representative experiment is shown of two independent experiments. One dot represents one cell. ***: significant (Mann Whitney U test, p<0.0001). Line indicates median + IQR.

https://doi.org/10.1371/journal.pone.0064029.g003

To evaluate the effects of docetaxel in vivo, H2B-D-expressing cells in the tumor were photo-marked and CFP-YFP ratios of single cells were subsequently determined before, 20 and 48 hours after a single intravenous docetaxel administration (25 mg/kg) (Figure 3B). This analysis revealed a significant increase in the CFP-YFP ratios of both C26 and SW480 tumor cells after docetaxel treatment when compared to pre-treated conditions (Figure 3B) and vehicle (PBS)-control (Figure S3A), indicating there is an increase in caspase-3 activity. Before docetaxel treatment, SW480 cells displayed a normalized CFP-YFP ratio of 1 on average, which increased to 2.26 and 2.34 after 20 and 48 hours of docetaxel treatment respectively. In line with these data, C26 cells also showed a significant increase in CFP-YFP ratio following docetaxel treatment (a ratio of 1.0 before and 1.09 and 1.61 after 20 and 48 hours of docetaxel treatment respectively) (Figure 3B).

To confirm our FRET results, we also performed fluorescence lifetime imaging microscopy (FLIM) (Figure 3C). Caspase-3 activation results in loss of FRET due to cleavage of the DEVD motif (Figure 2A) and therefore should cause an increase in the CFP fluorescence lifetime [31]. In line with our CFP-YFP ratio measurements, FLIM analysis of C26 cells after 25 mg/kg docetaxel treatment revealed a significant increase in CFP lifetime from, on average, 1.4 ns before docetaxel treatment to 1.7 ns at two days after docetaxel treatment (Figure 3C and D). The observed increase in caspase-3 activity was not caused by insertion of the imaging window or by light exposure during imaging, since FLIM analysis of tumor sections from within the tumor (not reached by either the window or the laser beam) showed that FRET was even more frequently lost in these areas (Figure S3B). In addition, in vivo FLIM analysis showed that CFP fluorescence lifetime was not increased upon vehicle (PBS) treatment (Figure S3C), confirming that both imaging and vehicle (PBS) treatment alone do not induce caspase-3 activity.

Together, these data show that caspase-3 activity, and therefore the onset of apoptosis, is induced upon treatment with docetaxel both in vitro and in vivo.

Docetaxel-induced apoptosis depends on the mitotic status of tumor cells in vitro but not in vivo

Next, we followed the effect of docetaxel treatment on mitotic progression using time-lapse imaging of both C26 and SW480 cells expressing H2B-D and the caspase-3 FRET probe (Figure 4). SW480 cells have a robust mitotic checkpoint and delay mitosis upon treatment with spindle poisons for at least 18 hours [32]. Indeed we find that docetaxel treatment in vitro resulted in a mitotic delay of ∼10–20 hours (Figure 4A, S4A). Of those mitotic cells, 22% showed an increase in CFP-YFP ratio (gray lines in Figure 4A) and clear apoptotic morphology during this mitotic delay (Figure 4A). The increase in CFP-YFP ratio was due to an increase in caspase-3 activity, since treatment with a pan-caspase inhibitor (z-VAD-fmk) abolished the docetaxel-induced increase in the number of cells that have an apoptotic CFP-YFP ratio (Figure S4B). Treatment of interphase cells with docetaxel in vitro did not affect CFP-YFP measurements (Figure S4C), which indicates that the increase in CFP-YFP ratio is specific for mitotically delayed cells. In line with previous data [9], [10], simultaneous imaging of apoptosis onset and mitotic progression shows that in vitro docetaxel treatment kills mitotic, but not interphase cells.

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Figure 4. Docetaxel induces apoptosis in vivo independent of a mitotic delay.

A, representative stills of SW480 cells in vitro entering mitosis in the presence of 1 µM docetaxel resulting in a mitotic delay (top) and eventually apoptosis (bottom). Bottom graph shows the CFP-YFP ratios of treated SW480 cells over time. Cells in which the CFP-YFP ratio reaches the apoptotic CFP-YFP ratio (>1.3 times increase) are indicated with gray lines and all other cells with black lines. B, left: Images of a mitotic C26 cell in vivo on day 2 after 25 mg/kg docetaxel treatment. Right: Quantification of the number of mitotic cells found at indicated time points (before, 20 h (day1) or 48 h (day2) after a single administration of 25 mg/kg docetaxel) in C26 tumors. Mitotic cells were determined according to nuclear morphology. C, quantification of the cell cycle state (mitotic or interphase) of cells with a docetaxel-induced apoptotic CFP-YFP ratio. The calculation of apoptotic CFP-YFP ratios (1.3 times increase) was performed as described in the Materials and Methods section. Data are plotted for both in vitro and in vivo measurements (n>20 cells per condition).

https://doi.org/10.1371/journal.pone.0064029.g004

To assess the mitotic response to docetaxel in vivo, we analyzed the number of mitotic cells following docetaxel treatment in both C26 and SW480 tumors using intravital imaging (Figure 4B). Strikingly, we have never observed any mitotic cells in SW480 cells during the 3 hour intravital imaging sessions (data not shown). For C26 cells, only a small percentage of mitotic cells were observed in C26 tumors both before and 20 hours after docetaxel treatment (1,4% and 1,2% respectively), which slightly increased at forty-eight hours after docetaxel treatment to 3.8% (Figure 4B). By contrast, analysis of C26 tumor slides stained for the mitotic marker phospho-Histone H3 revealed that the number of cells with mitotic figures is not increased in docetaxel-treated tumors when compared to vehicle (PBS)-treated tumors (Figure S5A and B). Thus, the small increase of mitotic C26 cells in vivo upon docetaxel is not significant or very small.

The data above shows that docetaxel treatment in vivo does not lead to a clear increase in the number of mitotic cells, which suggest that for most cells, apoptosis is not mediated through a delay in mitosis. Nevertheless, if docetaxel-induced apoptosis is mediated through mitotic delay, cells should simultaneously show a continuous apoptotic CFP-YFP ratio and stay in mitotis/tetraploid state until they fragment. To test this, we analyzed the ratio of mitosis and interphase only in cells in which apoptosis (apoptotic CFP-YFP ratio) is induced by docetaxel. This analysis showed in vivo that less than 7% of all SW480 and C26 cells in which apoptosis is induced were in mitosis (Figure 4C). Since cells with mitotic delay should stay in mitosis until they fragment, the latter data shows that of all docetaxel-induced apoptosis (apoptotic CFP-YFP ratio), at most 7% is induced by mitotic delay and at least 93% by mitotic-independent mechanisms. In contrast, the percentage of mitotic cells of all apoptotic SW480 and C26 cells in vitro was 75% and 60% respectively (Figure 4C). The latter data suggest, contrary to the in vivo situation, that the contribution of a delayed mitotic progression to docetaxel-induced apoptosis in vitro is very large (Figure 4C).

Potential mechanisms that explain the lack of induction of apoptotic delay by docetaxel

Two possible explanations for the absence of a clear increase in the number of mitotic cells in vivo are either that only low docetaxel concentrations are able to reach the tumor or that we miss mitotically delayed cells due to the relatively short imaging sessions (3 hours). Additionally, docetaxel could have a relatively short half-life in vivo, leading to different effects in vivo compared to the in vitro situation. To address these hypotheses, we analyzed the number of cells with an abnormal nucleus in vitro and in vivo, which is indicative of earlier mitotic defects (Figure 5A,B). Both mitotic cells treated with low doses of taxanes [13] and cells that slip out of a taxane-induced prolonged mitosis [10] undergo chromosome segregation errors, which lead to abnormal nuclei formation upon mitotic exit. Indeed, we find that low doses of docetaxel treatment in vitro induced the formation of abnormal nuclei and apoptosis in both C26 and SW480 cells (Figure 5A, S4A). However, in vivo analysis did not reveal striking differences before or after docetaxel treatment (Figure 5B, S6A). C26 tumors showed a slight increase in cells with abnormal nuclei after docetaxel treatment (12,6% before and 15,5% and 15,1% at 20 and 48 hours after docetaxel treatment respectively), but this increase did not correlate with the substantial increase in the percentage of apoptotic cells observed in the same fields (10%,14% and 70% before, 20 and 48 hours after docetaxel treatment respectively) (Figure 5B). The basal number of SW480 cells with abnormal nuclei was higher than that of C26 cells (Figure 5A,B). However, SW480 cells are intrinsically chromosome unstable, which means that they frequently lose and gain whole chromosomes during cell division, already in the absence of any treatment [33], [34]. Importantly, the number of nuclei in vivo with an aberrant morphology (Figure 5B) did not increase over time upon docetaxel treatment (34% and 25% of cells had an abnormal nucleus 20 and 48 hours after treatment respectively, compared to 27% before treatment). This indicates that these cells did not undergo chromosome segregation errors upon docetaxel treatment in vivo, whereas the percentage of apoptotic cells increased substantially in the same tumor fields (20%, 49% and 58% before, 20 and 48 hours after docetaxel treatment respectively) (Figure 5B, and see Figure S6B for the vehicle (PBS)-control). We also find that the number of photo-marked SW480 cells in vivo did not increase over time in docetaxel-treated tumors (Figure 5C), which provides additional proof that these cells have not divided in between the different imaging sessions. To address the hypothesis that the differences between our in vitro and in vivo data may be due to a relatively short half-life of docetaxel in vivo, we performed an in vitro docetaxel washout experiment (Figure S6C) to mimic a short exposure to docetaxel. This experiment showed that a short exposure to docetaxel still results in mitotic defects in vitro. Both SW480 and C26 cells that entered mitosis after docetaxel washout remained in mitosis for several hours (Figure S6C). These data indicate that docetaxel cannot be properly washed out in vitro and therefore most likely does not have a short half-life in tumor cells in vivo either. In addition, we observed an increase in mitotic cells with chromosome alignment defects in vivo upon docetaxel treatment, indicating that the drug did enter the tumors sufficiently long to affect mitotic progression in these cells (Figure S5C). The differences in the mode of apoptosis induction between in vivo and in vitro can therefore not be explained by a short half-life of docetaxel.

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Figure 5. Docetaxel induces apoptosis in vivo independent of mitotic defects.

A, left: Representative images of nuclei (DAPI) of C26 and SW480 cells in vitro after 1 day treatment with or without a low dose of docetaxel (2 nM). Scale bars represent 10 µm. Right: The number of C26 or SW480 cells with abnormal nuclei in vitro after indicated conditions. B, left: Representative images of SW480 cells in vivo with normal or abnormal nuclear morphology visualized using H2B-D. Scale bars represent 10 µm. Right: The number of cells with abnormal nuclei (black, see Figure S6A for representative images) and number cells with an apoptotic CFP-YFP ratio (>1.3 times increase) (gray) in SW480 and C26 tumors plotted at indicated time points (before, one day or two day after single intravenous administration of 25 mg/kg docetaxel). The n indicates number of cells analyzed. C, left: Representative images of photo-marked SW480 cells in vivo switched before docetaxel treatment and imaged on day 1 and 2 after single treatment. Scale bar represents 50 µm. Right: Quantification of the number of photo-marked (red) H2B-D cells in SW480 tumors in vivo. Relative number compared to day 0 (before treatment) is indicated. Average + SD is shown of 3 independent positions.

https://doi.org/10.1371/journal.pone.0064029.g005

Taken together, these data show that the onset of docetaxel-induced apoptosis correlates with the number of mitotic defects in vitro, but not in vivo. From this we conclude that docetaxel, in contrast to its effect in vitro, can induce apoptosis in vivo independent of its effect on mitotic progression.

Ethics statement

All mouse experiments were approved by the animal ethical committee (DEC) of the Netherlands Academy of Sciences (KNAW), the Netherlands, and animals were kept at the Hubrecht animal facility in Utrecht, the Netherlands. All surgeries and imaging sessions were performed under anesthesia using isoflurane and imaging sessions were limited to 3 hours per day to minimize suffering.

Cell culture, cell lines and reagents

SW480 cells were obtained from the cryobank of the University Medical Center Utrecht and were previously described [43]. C26 cells were a kind gift of O. Kranenburg and were previously published [45]. Cells were grown in DMEM (Lonza), supplemented with 6% FCS (Clontech), pen/strep (Invitrogen) and ultraglutamine (Lonza). In vitro time lapse imaging of cells was performed in Leibovitz medium (Lonza) supplemented with 6% FCS, pen/strep and ultraglutamine. SW480 and C26 cell lines were infected with lentivirus carrying pLV.CMV.puro-c3-CAAX or pWPXLd.c3-CAAX respectively and pLV.CMV.puro.H2B-Dendra. Cell lines were selected with 2 µg/ml puromycin (after pLV.CMV.puro infection) and single colonies were selected after replating 1–2 cells/well or using FACS sorting. Puromycin and docetaxel (used in cell culture experiments) were from Sigma and zVAD-fmk (50 µM) was from Calbiochem.

Plasmids

The caspase-3-CAAX FRET probe [23], [26] was ligated in pLV.CMV.puro or pWPXL.d. pLV.CMV.puro was linearized using Pst1 (+blunt with Klenow) and subsequently cut with EcoR1. pWPXL.d was linearized using BamH1 (+blunt) and subsequently cut with EcoR1. Caspase-3 CAAX was obtained after restriction of pcDNA3.c3-CAAX with HindIII (+Blunt) and EcoR1. H2B-Dendra2 was ligated in pLV.CMV.puro. Enzymes were all from NEB. Both pLV.CMV.puro and pWPXL.d were a kind gift from Dr. Patrick Derksen.

Live cell imaging in vitro

Cells were plated in 8-well chambered glassbottom slides (LabTek) and imaged in a heated chamber at 37°C using either a Leica TCS SP5 AOBS two-photon microscope (Mannheim, Germany) or a DeltaVision RT system (Applied Precision) with a 20×/0.75NA objective (Olympus) using SoftWorx software. The stored images were analyzed using LasAF software or ImageJ. Unless indicated, docetaxel was added 10 minutes prior to filming. Fluorescent images were acquired every 30 minutes for a duration of 48 hours.

DTX washout experiment

Cells were incubated with 1 µM docetaxel for a period of two hours, followed by three PBS-washing steps. After the addition of fresh medium, cells were imaged in a heated chamber (37°C and 5% CO2) using a DeltaVision RT system (Applied Precision) with a 20×/0.75NA objective (Olympus) using SoftWorx software. Image analysis was performed using ImageJ.

Colony formation assay

Cells (∼50.000/well) were plated on 6-wells plates (Costar) (day 0). Docetaxel was added on day 1. On day 6, plates were washed with PBS, fixed for 5 minutes with 96% Methanol, stained with 0,1% crystal violet.dH20 and scanned for analysis. Quantification was performed using ImageJ image analysis.

Window surgery

Window was placed 2–6 weeks after injection of 0,5 –1 million SW480 or C26 cells stably expressing both H2B-Dendra and the caspase-3 FRET probe. All surgical procedures were performed under 2% isoflurane inhalation anesthesia and under aseptic conditions. Before surgery, the tumor area was shaved and the skin was disinfected using 70% EtOH. An incision was made through the skin, where the imaging window was inserted (for details see [30], [46]). The window was secured with a non-absorbable, non-woven purse-string suture (4-0 prolene suture). After surgery the mice were kept at 32°C until fully recovered from anesthesia. Mice were closely monitored for a few hours after surgery and food was provided within the cage.

Intravital imaging

Mice were sedated using isoflurane inhalation anesthesia (1.5% to 2% isoflurane/O₂ mixture), and placed with their head in a facemask within a custom designed imaging box. The imaging box and microscope were kept at 32°C using a climate chamber surrounding the complete microscope stage, including the objectives. Mice were imaged for a maximum period of 3 hours per day. The mouse vitals were monitored during imaging using the MouseOx system (starr lifescience Corp, Oakmont, PA, USA). Imaging was performed on an inverted Leica TCS SP5 AOBS two-photon microscope (Mannheim, Germany) with a chameleon Ti:Sapphire pumped Optical Parametric Oscillator (Coherent Inc. Santa Clare, CA, USA). Docetaxel obtained from the University Medical Center Utrecht pharmacy was diluted in PBS to a stock concentration of 5 mg/ml. A single dose of 5 µl per gram mouse was administered intravenously to obtain a concentration of 25 mg/kg in vivo. PBS was used as vehicle control treatment and was administered similarly to docetaxel.

Image analysis was performed using ImageJ software. CFP-YFP ratios of single cells were determined by drawing specific regions of interest (ROIs) around cells and dividing the CFP average intensity by the YFP average intensity. Background levels for CFP and YFP were subtracted prior to ratio calculation.

Caspase-3 CFP-YFP ratio imaging

CPF and YFP in vitro and in vivo images were acquired by only exciting CFP (in vitro: 405 nm, in vivo: 820 nm) as described above. Before every experiment, the PMT settings of very channel were optimized, but kept constant during the course of the multiple day experiment. The CFP-YFP ratio was analyzed using Leica TCS and ImageJ software. To compare different experiments and conditions, the relative CFP-YFP ratio was calculated by normalizing all ratios to the CFP-YFP ratio at the start of the experiment.

FLIM analysis

FLIM analysis was carried out using a Leica TCS SP5 inverted microscope (Mannheim, Germany) with a 25× (HCX IRAPO N.A.0.95 WD 2.5 mm) water objective, which was adapted for TCSPC (time-correlated single-photon counting) FLIM with a Becker and Hickl SPC 830 card using 64 time channels. The samples were excited using a femtosecond titanium chameleon Ti:Sapphire pumped Optical Parametric Oscillator (Coherent Inc. Santa Clare, CA, USA (80 MHz). Images were obtained with a line-scan speed of 400 Hz. Two-photon excitation was carried out using a wavelength of 820 nm and fluorescence was detected between 450 nm and 480 nm. The fluorescence decays obtained were fitted using a single exponential decay model with Becker and Hickl SPCImage software v2,9,9, 29107, and the lifetimes were portrayed in false color maps.

Isolation of tumors

Tissues were isolated and fixed in periodate-lysine-paraformaldehyde (PLP) buffer (2,5 ml 4% PFA+0,0212 g NaIO₄+3,75 ml L-Lysine+3,75 ml P-buffer (pH 7.4)) O/N at 4 °C. On the following day, the fixed tissues were washed twice with P-buffer and placed for at least 6 hours in 30% sucrose at 4 °C. The tissues were then embedded in OCT tissue freezing medium (Jung) and stored at −20°C before sectioning.

Phospho-Serine 10 Histone H3 staining (pHH3)

PLP tumor sections were pre-incubated with TBS-0,1% Tween20 4% BSA for 30 minutes at room temperature. Anti-phospho-Histone H3 (1∶250) was incubated for 3 hours in TBS-0,1% Tween20 4%BSA, washed twice with TBS-0,1% Tween20 and secondary antibody was incubated for 45 minutes in the presence of DAPI. Prolong Antifade was used to mount the slides and analysis was done on a DeltaVision RT system (Applied Precision) with 60× or 10× objective (Olympus) using SoftWorx software.

Cleaved capase-3 staining

PLP tumor sections were rehydrated in PBS for 10 minutes at room temperature, after which tissue was blocked for 30 minutes in PBS with 5% NGS, 2,5% BSA, and 0,3% Triton X-100. Anti-cleaved-caspase-3 (1∶200) was incubated overnight in 0,5× blocking solution, washed three times with PBS and secondary antibody was incubated for 2 hours in 0,5× diluted blocking solution. After washing with PBS, tissues were mounted with Vectashield hard set mounting medium (H-1400, Vector Laboratories Inc., Burlingame, CA). Analysis was performed on a Leica TCS SP5 AOBS inverted microscope (Mannheim, Germany) with a 25× (HCX IRAPO N.A.0.95 WD 2.5 mm) water objective.