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. 2013 Aug;15(8):863–874. doi: 10.1593/neo.13932

Noninvasive Monitoring of Pharmacodynamics and Kinetics of a Death Receptor 5 Antibody and Its Enhanced Apoptosis Induction in Sequential Application with Doxorubicin1

Thomas G Weber *, Thomas Pöschinger *, Stefanie Galbán †, Alnawaz Rehemtulla †, Werner Scheuer *
PMCID: PMC3730039  PMID: 23908588

Abstract

Induction of apoptosis plays a crucial role in the response of tumors to treatment. Thus, we investigated the pharmacodynamics and tumor saturation kinetics of a death receptor 5 antibody (anti-DR5) when combined with chemotherapeutics. For our investigations, we applied an imaging method that allows monitoring of apoptosis noninvasively in living mice. A stably transfected apoptosis reporter based on split luciferase technology facilitates to screen various chemotherapeutics and anti-DR5 on their ability to induce apoptosis in glioblastoma cells in vitro as well as in vivo. We found that doxorubicin (DOX) treatment in vitro led to significant apoptosis induction within 48 hours and to a 2.3-fold increased anti-DR5 binding to the cell surface. In contrast, cisplatin and 5-fluorouracil (5-FU) treatment altered anti-DR5 binding only marginally. Induction of apoptosis by treatment with anti-DR5 was dose- and time-dependent (both in vitro and in vivo). Simultaneous visualization of fluorescence-labeled anti-DR5 in tumor tissue and apoptosis revealed maximal apoptosis induction immediately after the compound had reached tumor site. Regarding combination therapy of anti-DR5 and DOX, we found that the sequential application of DOX before anti-DR5 resulted in synergistically enhanced apoptosis reporter activity. In striking contrast, anti-DR5 given before DOX did not lead to increased apoptosis induction. We suggest that DOX-induced recruitment of DR5 to the cell surface impacts the enhanced apoptotic effect that can be longitudinally monitored by apoptosis imaging. This study demonstrates that the combination of apoptosis and fluorescence imaging is an excellent method for optimizing dosing and treatment schedules in preclinical cancer models.

Introduction

The ability of cancer cells to evade apoptosis is one of the hallmarks of cancer [1]. To target cancer cells effectively, it is necessary to treat them with combinations of anticancer drugs that reinforce the programmed cell death through the well-characterized intrinsic and extrinsic apoptosis pathways [2].

The intrinsic apoptosis pathway can be activated by chemotherapeutic compounds that often lead to DNA damage and cell cycle arrest [3]. This pathway signals through caspase-9 and is highly regulated by p53 and numerous antiapoptotic [e.g., Bcl-2, survivin, X-linked inhibitor of apoptosis protein (XIAP)] and proapoptotic proteins.

The extrinsic apoptosis pathway is initiated after binding of, e.g., TNF-related apoptosis-inducing ligand (TRAIL) to its death domain-containing receptors (DR4 and 5). The activated death domain cleaves procaspase-8 to caspase-8 [4]. Another substrate of caspase-8 is the BH-3 interacting domain death agonist (Bid). Cleaved Bid triggers cytochrome c release out of mitochondria linking the extrinsic and intrinsic apoptosis pathway [5]. Both pathways end in the activation of the effector caspases-3/7 that ultimately cleave cellular proteins at specific aspartate-glutamate-valine-aspartate (DEVD) cleavage sequence for caspases-3/7 and cause apoptosis [6].

The death receptors (DRs) are attractive targets for anticancer treatments. Treatments of tumor cells and tumors in preclinical models with human recombinant TRAIL achieved impressive anticancer effects, but its application for anticancer treatment is limited because of resistance development to TRAIL and its short half-life in serum [7,8]. Activating monoclonal antibodies to DRs with longer half-lives have already been developed, e.g., drozitumab that is a human agonistic antibody to DR5 and directly induces apoptosis by DR5 clustering through the extrinsic pathway [9,10].

Monitoring apoptosis in vivo would lead to a better understanding of the pharmacodynamics of such proapoptotic compounds. Furthermore, dosing, scheduling, and combinations of these compounds can be refined in preclinical settings by aiming to maximize the apoptotic effect. Especially, the sequential application of anticancer drugs targeting different proliferation or apoptosis pathways showed promising enhanced apoptotic effects in some xenograft models [11,12]. The time-staggered scheduling could lead to a synergistic effect owing to a higher susceptibility of tumor cells for second-line treatment after pretreatment [13]. The potential of such scheduled combinations to enhance apoptosis could be assessed in more detail by monitoring apoptosis in animal studies over time.

Thus, we applied a bioluminescence-based method that allows highly sensitive and noninvasive monitoring of apoptosis in living mice during treatment [14,15]. We showed recently that bioluminescence imaging is a valuable and reliable technique for evaluation of drug efficacy [16]. For monitoring apoptosis by bioluminescence imaging, an apoptosis reporter construct is stably transfected in the tumor cell line of interest. This construct consists of two split luciferase (Luc) components (C-Luc and N-Luc) that are separated by a specific DEVD cleavage sequence for effector caspases-3/7 (Figure 1). Constitutive expression of the construct is achieved by an upstream EF1-α promoter. Upon caspase-3/7 activation in apoptotic cells, the expressed reporter constructs are cleaved at the DEVD site leading to the complementation of C-Luc and N-Luc to form a functional Luc enzyme. This bioluminescence-based method offers a high signal-to-noise ratio resulting in a highly sensitive approach for monitoring apoptosis during treatment in human cancer xenografts [17]. In addition, it allows a comprehensive monitoring of apoptosis because the detected effector caspases-3 and -7 are common players in the intrinsic as well as in the extrinsic apoptosis pathway (Figure 1).

Figure 1.

Figure 1

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Schematic illustration of the caspase-3/7 GloSensor reporter activation in apoptotic cells.

We implemented this apoptosis reporter system in a subcutaneous xenograft model of the human glioblastoma multiforme (GBM) cell line D54. This cancer type has high medical needs due to median survival times of approximately 1 year after diagnosis despite standard-of-care treatment with temozolomide and radiotherapy [18]. An important issue is to investigate alternative treatments and to improve therapy strategies in preclinical settings. Thus, we used the apoptosis reporter to monitor apoptotic cell death in subcutaneous GBM tumors through optimizing dosing, schedule, and combination therapies of compounds targeting the intrinsic apoptosis pathway [e.g., cisplatin and doxorubicin (DOX)] and the extrinsic pathway (anti-DR5 antibody).

Materials and Methods

Reagents and Anti-DR5 Generation

Anti-DR5 antibody, a fully human agonistic monoclonal antibody to DR5, was kindly provided by Roche Glycart AG (Schlieren, Switzerland). For generation of the monoclonal anti-DR5 antibody, the variable light and heavy chains were synthesized on the basis of the antibody sequences included in the patent application US 2007/0031414 A1 [19]. The variable genes were fused in frame with human IgG1 constant κ and constant heavy chain, respectively, in standard mammalian expression vectors. The anti-DR5 antibody was transiently produced in HEK293 EBNA cells and purified by standard Protein A chromatography followed by size exclusion chromatography. Anti-DR5 was labeled in-house with Alexa Fluor 750 (A750) by monoreactive N-hydroxysuccinimide ester for specific labeling of amine residues according to the manufacturer's instructions (Invitrogen, Hamburg, Germany). The chemotherapeutic DOX (Hexal AG, Holzkirchen, Germany) was used for in vivo and in vitro studies. In addition, staurosporine (Roche Diagnostics GmbH, Mannheim, Germany), rhTRAIL (R&D Systems, Minneapolis, MN), temozolomide (Sigma-Aldrich, St Louis, MO), docetaxel (Sanofi-Aventis, Paris, France), cisplatin, 5-fluorouracil (5-FU), and irinotecan (all Medac, Hamburg, Germany) were tested in vitro. d-Luciferin firefly potassium salt was purchased from Biosynth AG (Staad, Switzerland).

Cell Lines and Culture

D54-caspase-3/7 GloSensor cell line, developed by the Department of Radiation Oncology and Radiology, University of Michigan (Ann Arbor, MI) [15,17], was cultured in RPMI 1640 medium (PAN Biotech GmbH, Aidenbach, Germany) supplemented with 2mM l-glutamine, 10% FBS (both PAN Biotech GmbH), and 200 µg/ml G-418 (Roche Diagnostics GmbH).

Bioluminescence Assays

D54-caspase-3/7 GloSensor expressing cells (15,000 cells per well) were seeded in white clear bottom 96-well plates (PerkinElmer, Groningen, Netherlands) and incubated for 24 hours. Cells were treated with various compounds in different concentrations for 4, 24, and 48 hours. d-Luciferin (300 µg/ml) was added to each well and bioluminescence signals were measured 5 minutes after luciferin addition by Safire2 reader (Tecan Group Ltd, Männedorf, Switzerland). Sequential application was performed by adding the second drug 18 hours after first drug, and bioluminescence signals were measured 6 hours after first drug, 6 and 26 hours after second drug application. Subsequently, CellTiter-Glo solution (Promega, Madison, WI) was added to each well to determine cell viability according to the user's instructions. Bioluminescence signals were measured 5 minutes after CellTiter-Glo substrate addition by Safire2 reader. Interfering influences of both bioluminescence-based assays were excluded by control experiments. The signals of the apoptosis reporter assay were divided by CellTiter-Glo signals to minimize bias caused by lower living cell counts in treated wells. This ratio was divided by mean ratio of untreated wells at the distinct time points to get fold induction.

Homogeneous Caspase Assay

D54-caspase-3/7 GloSensor cells (15,000 cells per well) were seeded in black clear bottom 96-well plates (PerkinElmer) and incubated for 24 hours. Cells were treated with various compounds in different concentrations for 4, 24, and 48 hours. The Homogeneous Caspase Assay (Roche Diagnostics GmbH) was performed according to the user's instructions. Fluorescence signals were measured by Safire2 reader. Fluorescence signals were divided by CellTiter-Glo signals and mean ratio of untreated wells at 4, 24, or 48 hours.

Flow Cytometric Analysis

D54-caspase-3/7 GloSensor cells (1 x 106 per six-well plate) were left untreated or treated with 1 µg/ml anti-DR5, 1 µM DOX, 10 µM cisplatin, or 100 µM 5-FU, respectively, for 18 hours. Cells were harvested and washed in phosphate-buffered saline (PBS). After protein blocking for 15 minutes, 4 µg/ml anti-DR5-A750 diluted in BD Pharmingen Stain Buffer (BSA; BD Pharmingen, San Jose, CA) was incubated for 30 minutes on ice. Sodium azide (NaN3) in the Stain Buffer prevents anti-DR5-A750 from internalization. After washing twice in Stain Buffer, the cells were resuspended in Stain Buffer for fluorescence-activated cell sorting (FACS) analysis. Alternatively, cells were incubated (20 minutes, 4°C) with BD Cytofix/Cytoperm (BD Pharmingen) for permeabilization of the cell membrane before anti-DR5-A750 incubation. Washing steps and antibody dilution were then done with BD Perm/Wash Buffer (BD Pharmingen). Human IgG (Life Technologies, Darmstadt, Germany), in-house labeled with A750, was used as an isotype control. FACS was performed using MACSQuant Analyzer (Miltenyi, Bergisch Gladbach, Germany), and results were analyzed by FlowJo software (FlowJo, Ashland, OR).

Immunofluorescence Staining

D54-caspase-3/7 GloSensor cells (200,000 cells) were seeded on Millicell EZ slides (Millipore, Tullagreen, Ireland) and left untreated or treated with 1 µM DOX for 18 hours. Cells were then permeabilized with BD Cytofix/Cytoperm for 20 minutes at 4°C and washed twice with BD Perm/Wash Buffer before incubating 1 µg/ml anti-Golgin-97 antibody (Invitrogen, Eugene, OR) for 30 minutes at 4°C. After washing twice, a mixture of 5 µg/ml Hoechst 33342 (BD Pharmingen), 1 µg/ml Alexa 488-labeled Cholera toxin subunit B, 2 µg/ml Alexa Fluor 647 goat anti-mouse IgG1 (both Invitrogen), and 4 µg/ml anti-DR5-A750 was incubated for 30 minutes at 4°C. Slides were washed twice with PBS and analyzed by multispectral fluorescence microscopy using Pannoramic 250 1.14 slide scanner and Pannoramic Viewer 1.15 (3DHISTECH, Budapest, Hungary). For cell surface binding studies, anti-DR5-A750 (4 µg/ml) was incubated in BD Pharmingen Stain Buffer (BSA) for 30 minutes at 4°C before cell permeabilization.

Human Apoptosis Array

D54-caspase-3/7 GloSensor cells (1 x 106 cells per six-well plate) were left untreated or treated with 1 µg/ml anti-DR5, 1 µM DOX, the combination of both given simultaneously, anti-DR5 18 hours before DOX, or reverse. Cells were lysed 18 hours after single treatment or 24 hours after combination treatment, and Human Apoptosis Array Kit (R&D Systems) was performed according to the user's instructions. Briefly, 250 µg of protein was incubated overnight on each nitrocellulose membrane. After washing thrice, Detection Antibody Cocktail (diluted 1:100) was incubated for 1 hour, followed by washing thrice and incubation of streptavidin-HRP (diluted 1:2000) for 30 minutes. After another washing step, Chemi Reagent Mix was added and the signals on the membranes were developed. The pixel volume of the spot signals was quantified by ImageQuant TL (GE Healthcare, Little Chalfont, United Kingdom) and normalized to reference spots.

Western Blot Analysis

Cells were treated and lysed as described in the Human Apoptosis Array section. Protein solution (15 µg) was mixed with NuPAGE LDS Sample Buffer and NuPAGE Sample Reducing Agent, heated at 75°C for 10 minutes, and run on a NuPAGE 4% to 12% gradient sodium dodecyl sulfate gel (all Invitrogen, Carlsbad, CA). After the gel was blotted on a nitrocellulose membrane, the membrane was blocked in 1 x NET Buffer for 2 hours. Subsequently, 1 µg/ml rabbit anti-human Bid antibody (R&D Systems) was incubated for 2 hours at room temperature. After washing thrice in NET Buffer, anti-rabbit IgG HRP antibody (diluted 1:2000; Cell Signaling Technology, Dan-vers, MA) was incubated for 1 hour at room temperature. After washing thrice in NET Buffer, blots were developed. Rabbit polyclonal antibody to β-actin (diluted 1:4000; Abcam, Cambridge, United Kingdom) was used as a loading control and was incubated overnight.

Subcutaneous Xenograft Model

Female severe combined immunodeficient/beige (SCID beige) and SCID hairless outbred mice were obtained from Charles River (Sulzfeld, Germany) and were 9 to 12 weeks of age at initiation of experiments. Five million D54-caspase-3/7 GloSensor cells in 100 µl of PBS were inoculated in the right flank of each mouse under 2% isoflurane anesthesia. After the tumors had grown to approximately 200 to 250 mm3 in volume, the animals were randomized in therapy groups (five mice per group) according to their basal bioluminescence signal and their tumor volume. Treatment compounds were given intravenously through the tail vein of mice. Length (L) and width (W) of the tumors were measured by caliper everyday, and tumor volumes were calculated as (L x W 2)/2 with L ≥ W . All animal studies were approved by the local government (File No. 55.2-1-54-2532.2-26-09).

Bioluminescence Imaging

Mice were injected intraperitoneally with a single dose (100 µl) of d-luciferin (150 mg/kg in PBS) 10 minutes before measurement. Bioluminescence imaging was performed with IVIS spectrum (Caliper Life Sciences, Hopkinton, MA) under constant isoflurane (2%) anesthesia. The acquired bioluminescence signals in a region of interest (ROI) over the tumor site were read out as total flux radiance (p/sec/cm3/sr) with Living Image software (Caliper Life Sciences). Every ROI signal was divided by the related tumor volume and by the appropriate ratio of bioluminescence signal to tumor volume at start point 0 hour to get fold induction. At the application day of the drug, mice were measured three times followed by measurements every 1 to 3 days.

Fluorescence Imaging

Fluorescence imaging of mice treated with anti-DR5-A750 was performed with IVIS spectrum directly subsequent to bioluminescence imaging to allow co-localization of both readouts. “AF750” as filter setting was chosen in the Imaging Wizard controller of Living Image. Fluorescence signal intensities were determined by manual spectral unmixing of acquired images and ROI analysis over the tumor site. Spectrum of A750 was acquired by measuring anti-DR5-A750 solution in a black 96-well plate.

Necropsy and Histologic Analysis

Mice were sacrificed by transcervical dislocation. Subcutaneous tumors were explanted, fixated in 10% formalin, dehydrated, and blocked in paraffin. Paraffin sections (2.5 µm) were stained with human/mouse cleaved caspase-3 monoclonal antibody (R&D Systems, Wiesbaden, Germany) on a Discovery XT (Ventana Medical Systems, Tucson, AZ). Briefly, cleaved caspase-3 antibody was incubated for 60 minutes, followed by a washing step and incubation of UltraMap anti-rabbit HRP (Ventana Medical Systems) for 16 minutes. Finally, the sections were counterstained with hematoxylin. To determine the proliferation status of the tumors, paraffin sections were stained with CONFIRM anti-Ki-67 (clone 30-9) rabbit monoclonal antibody on a BenchMark XT (both Ventana Medical Systems). Slides were analyzed by bright-field microscopy using Pannoramic 250 1.14 slide scanner and Pannoramic Viewer 1.15 (3DHISTECH). Anti-DR5-A750 accumulation in tumor tissue was analyzed by multi-spectral fluorescence microscopy using Pannoramic 250 1.14 slide scanner and Pannoramic Viewer 1.15.

Statistical Analysis

All data values are represented as mean + SEM. Statistical analysis was performed using JMP8 software (SAS, Cary, NC). Two-sided t test was applied for in vitro studies with normally distributed values. For animal studies, the areas under the curve were calculated and Wilcoxon test was applied. P values < .05 were considered as statistically significant.

Results

Screening for Apoptosis-Inducing Compounds

To find appropriate apoptosis-inducing compounds for subsequent in vivo therapy studies, various chemotherapeutics and therapeutic antibodies were screened for their ability to activate the bioluminescence apoptosis reporter in vitro. D54-caspase-3/7 GloSensor expressing cells were treated with different concentrations of the compounds for 4, 24, and 48 hours, and bioluminescence signals were quantified (Figure 2A). Anti-DR5-treated cells showed up to 38-fold induction in signal intensities within the first 4 hours compared to untreated cells. TRAIL strongly activated the apoptosis reporter (up to 29-fold induction) and showed similar dynamics compared to anti-DR5. Chemotherapeutics DOX (1 µM) and cisplatin (100 µM) caused maximal effects with six- and nine-fold induction, respectively, only after 48 hours. The standard-of-care chemotherapeutic for GBM therapy, temozolomide, led to a moderate 3.5-fold increase after 24 to 48 hours. Other treatments (e.g., 5-FU and irinotecan) did not show any significant increase in signal intensities.

Figure 2.

Figure 2

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In vitro evaluation of bioluminescence apoptosis reporter in D54 tumor cells. (A) Screening with various anticancer compounds in different concentrations for 4, 24, and 48 hours of incubation on their ability to activate the apoptosis reporter. Staurosporine (STS) served as a positive control. Bioluminescence signals were normalized to cell viability and expressed in fold induction compared to untreated samples; n = 4. (B) Fluorescence-based Homogeneous Caspase Assay was performed for a subset of treatments. Fluorescence signals were normalized to cell viability and expressed in fold induction compared to untreated samples; n = 3. (C) Correlation analysis of apoptosis reporter activities to Homogeneous Caspase Assay activities.

To verify the induction of the apoptosis reporter activity, the fluorescence-based Homogeneous Caspase Assay was performed for a subset of compounds (Figure 2B). This assay, which measures caspase-3/7 activity through cleavage of DEVD-Rhodamine 110 to free, fluorogenic Rhodamine 110, confirmed the increased bioluminescence signals due to caspase-3/7 activation with good correlations (coefficient of determination R2 =0.79; Figure 2C).

Dosage Optimization of Anti-DR5

To evaluate the bioluminescence apoptosis reporter in vivo, SCID beige mice bearing subcutaneous D54-caspase-3/7 GloSensor tumors were treated with two different doses of anti-DR5 (1 and 3 mg/kg). Mice receiving higher dose showed maximal tumor bioluminescence signals with 66-fold increase within 6 hours after treatment, whereas low-dose treatment led to up to 31-fold induction compared to vehicle (Figure 3, A and B). After 24 hours, the signals declined below 30-fold induction. The higher dose (3 mg/kg) kept the signals between 23- and 31-fold induction over 7 days, whereas the apoptosis reporter activity in tumors of mice treated with the 1 mg/kg dose decreased to 15- to 23-fold induction. After 8 days, both treatment groups showed considerably lower apoptosis reporter activities, indicating an appropriate time point for re-dosing. Re-application led instantly to increased reporter activities. Considering the whole treatment period, apoptosis induction by high-dose treatment is statistical significant compared to low-dose treatment (P < .02), and both dosages of anti-DR5 are highly significant compared to vehicle (P < .01). Immunohistochemical analysis of explanted tumor tissues at the end point verified increased active caspase-3 levels in anti-DR5-treated tumors (Figure 3C).

Figure 3.

Figure 3

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Dosage and application optimization of anti-DR5 in vivo. (A) Time course of bioluminescence apoptosis reporter activities in untreated mice and mice treated with 1 or 3 mg/kg anti-DR5, respectively. Second treatment was applied 8 days (192 hours) after first treatment; n = 5. (B) Representative bioluminescence images of untreated mice and mice treated with 1 or 3 mg/kg anti-DR5 12 hours after first treatment. Signals are expressed in radiance (p/sec/cm3/sr). (C) Representative immunohistochemical sections stained on active caspase-3 (brown) at end point. Cell nuclei were counterstained with hematoxylin (blue). Original magnification, x400.

Simultaneous Apoptosis and Fluorescence Imaging

Next, we wanted to investigate the mechanism of action of anti-DR5 in detail by combining pharmacodynamics with tumor saturation kinetics. Treatment of mice with 3 mg/kg A750-labeled anti-DR5 allowed additional kinetics studies of antibody accumulation in the tumor through fluorescence imaging. The highest fluorescence signals were already detected 6 hours after initiation of treatment, whereas the maximal apoptosis induction, measured by bioluminescence imaging, was evoked slightly time-shifted at 12 hours after antibody application. In contrast, maximal tumor growth inhibition was reached only after 5 days (Figure 4A). This emphasized the fast apoptosis induction through the extrinsic pathway and the lagged efficacy on tumor growth after initiation of programmed cell death. Over time, the antibody dissociated from the tumor with 50% dissociation after 3 days, whereas the apoptosis reporter activity was steadily elevated for more than 8 days after an initial activity peak. Successful reapplication of anti-DR5 was monitored by reconstituted accumulation of the fluorescence-labeled antibody in the tumor resulting instantly to increased apoptosis reporter activities and time-lagged, reinforced tumor growth inhibition. Co-localization of bioluminescence signals with subsequently acquired fluorescence signals demonstrated the binding of anti-DR5-A750 to D54-caspase-3/7 GloSensor tumor cells in vivo (Figure 4B). Compared to unlabeled anti-DR5, A750-labeled anti-DR5 showed almost identical pharmacodynamics (Figures 3 and 4).

Figure 4.

Figure 4

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Pharmacodynamics and tumor saturation kinetics after anti-DR5-A750 application. (A) Time courses of mean bioluminescence apoptosis reporter activities, mean fluorescence signals (anti-DR5-A750 accumulation in the tumor), and mean tumor growth inhibition (compared to untreated control group). Fluorescence signals were maximal at 6 hours, bioluminescence signals after 12 hours, and tumor growth inhibition after 5 days. (B) Representative bioluminescence, fluorescence, and overlay images of mice treated with 3 mg/kg anti-DR5-A750 12 hours after first treatment; n =5.

Combination of Anti-DR5 with Chemotherapeutics

Numerous in vitro and in vivo data have been published demonstrating that the combined application of two anticancer compounds enhances apoptosis [11,13,20]. Especially, the sequential application of two drugs that target different proliferation or apoptosis pathways-seems to be promising [21].

Thus, we investigated apoptosis reporter activities after sequential treatment of D54-caspase-3/7 GloSensor cells with anti-DR5 and DOX, cisplatin, or 5-FU, respectively. DOX and cisplatin alone induced apoptosis after 24 to 48 hours, whereas 5-FU did not show any apoptosis induction alone (see Figure 2). The in vitro apoptosis reporter experiments revealed a 54-fold increase of the apoptosis reporter signals when DOX was added 18 hours before anti-DR5 (DOX→ anti-DR5). Reverse (anti-DR5→DOX) or simultaneous (anti-DR5 + DOX) application with maximal 33- and 40-fold induction, respectively, showed only additive or slightly synergistic effects compared to anti-DR5 and DOX monotherapies with 22- and 9-fold induction, respectively (Figure 5A). Sequential application of cisplatin and anti-DR5 did not lead to any improved apoptotic effects over simultaneous dosing, which already led to an impressive synergism with up to 52-fold induction. Combination of anti-DR5 with 5-FU did not show any superiority over anti-DR5 monotherapy at all. Comparable results were obtained when first drug was given 24 hours instead of 18 hours before second drug. However, shorter (4 hours) or longer (48 and 72 hours) time intervals between DOX and anti-DR5 treatment showed significantly diminished apoptotic effects, indicating an optimized treatment schedule when DOX is given 18 to 24 hours before anti-DR5 (Figure 5B).

Figure 5.

Figure 5

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Effects of chemotherapy pretreatment on apoptosis induction and DR5 expression. (A) In vitro bioluminescence apoptosis reporter assay to validate apoptosis induction for sequential application of 1 µMDOX, 10 µMcisplatin, or 100 µM5-FU- and 1 µg/ml anti-DR5 compared to single and simultaneous treatment. In sequential treatments, second drug was added 18 hours after first drug treatment. Bioluminescence signals were normalized to cell viability and expressed in fold induction compared to untreated samples; n = 4; **P < .01.(B) In vitro bioluminescence apoptosis reporter assay showing apoptosis induction after different time intervals between DOX and anti-DR5 treatment. Apoptosis reporter activities were measured 6 hours after addition of second-line given anti-DR5. (C) FACS analysis of anti-DR5-A750 binding to cell surface of D54-caspase-3/7 GloSensor cells after no pretreatment or 18-hour pretreatment with 1 µMDOX, 10 µM cisplatin, or 100 µM 5-FU. (D) FACS analysis of anti-DR5-A750 binding to permeabilized D54-caspase-3/7 GloSensor cells after no pretreatment or 18-hour pretreatment with 1 µMDOX, 10 µM cisplatin, or 100 µM 5-FU. Cells were permeabilized before anti-DR5-A750 staining to allow intracellular binding of the antibody. (E) Immunofluorescence staining for anti-DR5 binding on the cell surface and in the cytosol of untreated or DOX-treated D54-caspase-3/7 GloSensor cells. The cells were permeabilized for intracellular binding studies. In untreated cells, anti-DR5-A750 signals (yellow) were rarely detected on the cell surface (Cholera toxin-A488 staining, green) but frequently detected on intracellular Golgi networks (Golgin-97/Alexa 647 antibody, magenta). In DOX-treated cells, anti-DR5-A750 signals were intensified on the cell surfaces. Blue, Hoechst cell nucleus staining; arrows, anti-DR5-A750 binding to the cell surface. Original magnification, x2500.

Guo et al. have shown that the enhanced apoptotic effect of DOX and TRAIL given sequentially in a glioblastoma xenograft model is due to an up-regulation of DR5 after DOX treatment [22]. Thus, we examined by FACS analysis whether pretreatment with DOX, cisplatin, or 5-FU causes an increased binding of anti-DR5 on the tumor cell surface. Results of FACS studies revealed that DOX treatment for 18 hours caused a 2.3-fold higher fluorescence signal intensity compared to untreated cells. This indicates that DR5 receptors were upregulated on the cell surface (Figure 5C). Cisplatin and 5-FU treatment led to an only moderate increase in signal intensities (1.6-fold). Almost identical results were obtained when FACS analysis was performed with A750-labeled mouse anti-human TRAIL R2/TNFRSF10B antibody (MAB6311; data not shown). In contrast, when cells were permeabilized before anti-DR5-A750 incubation to investigate total DR5 protein levels, FACS analysis revealed almost no differences in signal intensities of untreated cells and cells pretreated with DOX, cisplatin, or 5-FU (Figure 5D). The almost unchanged expression levels of total DR5 after treatment were confirmed by Western blot analysis (data not shown). This discrepancy between surface expression and total protein expression of DR5 assumes a quantitative domination of intracellular DR5 protein over surface-located DR5 receptor, which might explain similar total DR5 protein levels.

To visualize possible differences in intracellular and surface binding of anti-DR5 in untreated or DOX-treated cells, immunofluorescence staining and microscopy were performed (Figure 5E). Zhang et al. found DR5 receptors located in the trans-golgi network (TGN) of human melanoma [23]. We have been able to confirm this observation with the D54-caspase-3/7 GloSensor GBM cells using fluorescence staining of Golgi networks and co-localization with anti-DR5-A750 signals. Compared to untreated cells, we observed increased binding of anti-DR5-A750 to the cell surface membrane in the DOX-treated cells. This indicates that DOX induces translocation of DR5 from cytoplasm to the tumor cell surface. In concordance with this hypothesis, a shift of anti-DR5-A750 binding from Golgi structures to cell surface was detected in permeabilized cells after DOX treatment (Figure 5E).

Expression Profiles after DOX Treatment

To investigate the influence of DOX or anti-DR5 pretreatment in more detail, the expression profile of 35 apoptosis-related proteins was examined using the Human Apoptosis Array from R&D Systems (Figure 6A). The levels of active caspase-3 proteins were concordant with apoptosis reporter signal intensities for all tested treatments. Consistent with analysis described above, DOX pretreatment did not lead to an up-regulation of total DR5 protein levels (Figure 6B). High levels of phosphorylated p53 were seen in DOX-treated cell lysates, whereas DOX→anti-DR5 and anti-DR5 + DOX treated cells showed a decrease in antiapoptotic Bad, Bcl-2, XIAP, and survivin but also in proapoptotic HTRA2/Omi and SMAC/Diablo levels (Figure 6B). The most obvious difference between DOX→anti-DR5 and anti-DR5 + DOX in the Apoptosis Array could be found for cytochrome c levels, where DOX→anti-DR5 led to significant lower cytochrome c levels. In addition, protein levels of proapoptotic Bid were analyzed by Western blot analysis. Full-length Bid levels were significantly decreased in DOX→anti-DR5 and anti-DR5 + DOX treated cells, whereas a truncated, approximately 10 kDa form of Bid was increased (Figure 6C).

Figure 6.

Figure 6

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Expression level analysis of apoptosis-related proteins. (A) Human apoptosis array (R&D Systems) showing changes in protein expression levels of 35 apoptosis-related proteins in duplicates after anti-DR5/DOX treatment. (B) Quantification of protein expression of 13 apoptosis-related proteins of interest. The selected proteins are numbered and their locations on the array are marked in A. Mean pixel volumes of the duplicates were normalized to reference spots on the array. (C) Western blot analysis for Bid protein expression levels. Bid antibody detected full-length Bid (upper band, 22 kDa) and truncated Bid (tBid, lower band, 10 kDa). β-Actin served as a loading control.

Sequential Application of Anti-DR5 and DOX In Vivo

To verify the superiority of sequential DOX→anti-DR5 versus reverse and simultaneous treatment in vivo, SCID hairless outbred mice bearing subcutaneous D54-caspase-3/7 GloSensor tumors were treated with 1 mg/kg anti-DR5-A750 and 10 mg/kg DOX either simultaneously or sequentially. A time interval of 18 hours, which was determined in Figure 5B to be the optimal for maximal apoptosis induction, was chosen. Apoptosis induction monitored by bioluminescence imaging allowed quantifying differences in apoptotic responses for each application regimen. Fluorescence imaging should highlight increased anti-DR5-A750 binding to tumor tissues after DOX pretreatment. When anti-DR5-A750 was given 18 hours after DOX, an immediate activation of the apoptosis reporter with up to 35-fold induction and significant elevated activities over the study period of 6 days were observed (Figure 7A). Simultaneous or reverse application, however, showed only moderate apoptosis induction (maximal 7- to 12-fold), which was comparable to apoptosis induction mediated by anti-DR5-A750 monotherapy. The apoptosis reporter activities of anti-DR5 + DOX and anti-DR5→DOX treated mice almost returned to control level after 6 days. Induction of apoptosis by DOX treatment alone was delayed and peaked at day 6 with a moderate five-fold induction (Figure 7A).

Figure 7.

Figure 7

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Sequential application of 1 mg/kg anti-DR5-A750 and 10 mg/kg DOX. (A) Time course of mean bioluminescence apoptosis reporter activities in control mice and mice treated with anti-DR5-A750 + DOX, anti-DR5-A750→DOX, and DOX→anti-DR5-A750. Second drug was given 18 hours after first drug application. Reporter activities were normalized to tumor volume and expressed in fold induction to initial time point; n = 5. (B) Time course of mean fluorescence signal intensities showing the accumulation of anti-DR5-A750 in the tumor site after sequential application of anti-DR5-A750 and DOX. Signals were expressed in percent of mean maximal signal; n =5. (C) Changes in tumor volume caused by different sequential application of anti-DR5-A750 and DOX. Mean end point tumor volumes of each group (5 days after treatment initiation) were calculated and divided by initial mean tumor volumes; n = 5. (D) Representative histologic sections showing active caspase-3 levels, anti-DR5-A750 accumulation, and Ki-67 proliferation status in tumors explanted 24 hours after first drug application. All fluorescence slides were scanned with the same exposure time (t = 675 ms) to compare images. Original magnification, x400.

Regarding the different application schedules, fluorescence imaging using anti-DR5-A750 revealed differences in antibody accumulation in the tumor region. DOX→anti-DR5-A750 treatment resulted in significantly enhanced fluorescence signal intensities 6 hours after anti-DR5 application compared to anti-DR5-A750 + DOX treatment, suggesting a higher anti-DR5-A750 binding due to increased expression of DR5 on DOX pretreated tumor cells (Figure 7B). However, no significant difference becomes detectable at later time points and in comparison to anti-DR5-A750→ DOX treatment (Figure 7B). Excluding untreated and anti-DR5-treated mice, no significant differences in changes of tumor volume for the different application schedules were reached (Figure 7C). This indicates that apoptosis induction does not immediately translate to tumor volume reduction.

Ex vivo active caspase-3 staining on tumor tissues explanted 24 hours after first drug application confirmed the results of the in vivo apoptosis imaging. DOX→anti-DR5-A750 treated tumors revealed strongly enhanced caspase-3 activities, whereas anti-DR5-A750 + DOX and anti-DR5-A750→DOX treated tumors showed only moderate increase in caspase-3 activities compared to untreated controls (Figure 7D, left). Histologic detection of anti-DR5-A750 by fluorescence microscopy also confirmed the in vivo fluorescence imaging data. Anti-DR5-A750 bound to DOX→anti-DR5-A750 treated tumor cells more efficiently, again indicating an up-regulation of DR5 (Figure 7D, middle). Interestingly, Ki-67 proliferation staining revealed no impact on proliferation rate after anti-DR5 monotherapy, whereas DOX as well as DOX + anti-DR5 and DOX→anti-DR5 treatment inhibit proliferation of GBM tumor cells (Figure 7D, right). This suggests that, besides DOX-induced up-regulation of DR5, the proliferation inhibitory effect of DOX might also contribute to the superiority of DOX before anti-DR5 treatment.

Discussion

Here, we successfully applied the bioluminescence apoptosis reporter system in a preclinical model of GBM for monitoring pharmaco-dynamics of anti-DR5 and the enhanced apoptosis induction in a sequentially given therapy. The apoptosis induction quantified by the split Luc system correlated with the induction of caspase-3/7 activity (as shown by ex vivo histologic staining), which activates the Luc light reaction. The labeling of anti-DR5 with a fluorophore helped to investigate the mechanism of action of anti-DR5 on the extrinsic apoptosis pathway in more detail and allowed to refine the scheduling of this antibody.

Combined tumor saturation kinetics and pharmacodynamics revealed fast apoptosis induction after anti-DR5 reached the tumor site. The initial peak of apoptosis reporter signals can be explained by contemporaneous apoptosis induction in all tumor cells that are initially sensitive to anti-DR5 treatment. These cells rapidly abandon protein synthesis and apoptosis reporter expression, in particular, which leads to the decline of bioluminescence signals after approximately 16 to 24 hours [24]. Re-application of anti-DR5 reinforced bioluminescence signal intensities, but less prominent, indicating a smaller group of cells responding. These observations demonstrate the immediate activation of the extrinsic apoptosis pathway by anti-DR5 in vivo. Apoptosis induction by DOX treatment, however, started to occur only after 48 to 72 hours. This time-shifted activation of the two apoptosis pathways is accounted by the more complex intrinsic activation cascade [2].

Agonistic antibodies to DR5 as well as TRAIL showed promising antitumor effects in xenograft models [9,25,26] but lack efficacy in clinics [27–29]. One reason for the lack of efficacy is the development of resistance to DR5-dependent monotherapy [26,30]. Therefore, we investigated the potential of sequential application of anti-DR5 and DOX by apoptosis imaging. The drug partner DOX was prioritized on the basis of the split Luc assay performed in vitro. As shown in Figure 2A, DOX induced a concentration- and time-dependent induction of apoptosis and results depicted in Figure 5 indicate that the apoptosis induction depends on the application schedule of both drugs. These in vitro observations were confirmed in in vivo studies. Enhanced apoptosis after sequential application of TRAIL and DOX has previously been demonstrated in xenograft models of sarcoma, breast cancer, glioblastoma, myeloma, and prostate cancer [11,21,22,31,32]. However, these investigations only relied on efficacy or survival studies and ex vivo readouts but not on longitudinal apoptosis detection.

On the basis of our in vitro and in vivo results, the sequential application of DOX→anti-DR5 with a time interval of 18 to 24 hours is superior to anti-DR5 + DOX and anti-DR5→DOX indicating a synergistic effect when DNA damages are induced first. As previously shown, DOX pretreatment can sensitize cells making them more susceptible for TRAIL-mediated apoptosis [33]. Guo et al. showed that a possible explanation for the sensitization of GBM cells after DOX treatment might be the up-regulation of total DR5 and DR5 on the cell surface [22]. An up-regulation of DR5 is also described after treatments with other chemotherapeutics such as cisplatin, proteasome and histone deacetylase inhibitors, and natural products [34–36]. Figure 5A shows that the combination of cisplatin and anti-DR5 enhances apoptosis induction best when both compounds are given simultaneously. FACS analyses revealed that cisplatin pretreatment does not affect DR5 up-regulation on the cell surface in the way DOX pretreatment does. This might be one reason that the beneficial effect of cisplatin given before anti-DR5 is not as prominent as after DOX pretreatment. Therefore, the synergistic effect of combined cisplatin and anti-DR5 treatment might rely on the manipulation of different proapoptotic and antiapoptotic components such as Bid cleavage or down-regulation of antiapoptotic p27 and Bcl-x [36,37]. These manipulations might deploy their proapoptotic effects best when cisplatin and anti-DR5 are given simultaneously.

Besides an inhibition in tumor cell proliferation caused by DOX pretreatment, we confirmed DR5 up-regulation and increased anti-DR5 binding on the cell surface after DOX treatment by FACS analyses. For the first time, we proved this effect to DOX treatment in vivo by monitoring anti-DR5-A750 binding to the tumor. Pretreatment with DOX led to a higher anti-DR5-A750 accumulation in the tumor compared to simultaneous application. Guo et al. and Sheikh et al. could also detect an up-regulation in total DR5 protein expression for different tumor cell lines [22,38]. In contrast, our analyses demonstrate that DOX treatment does not modulate total DR5 protein levels. Bagci-Onder et al. explained such a discrepancy between cell surface and total DR5 expression with low endogenous DR5 receptor expression on the cell surface [28]. Because of low DR5 surface expression especially on untreated cells, which we showed by immunofluorescence microscopy, we suggest that intracellular DR5 proteins predominate DR5 receptors on the cell surface. The cellular and molecular mechanisms of the translocation process from intracellular DR5 proteins to the cell surface mediated by DOX treatment need to be further investigated.

Besides DR5 surface up-regulation, further prominent changes in expression levels of apoptosis-related proteins after DOX treatment are depicted in Figure 6C. The enhanced apoptotic effect of DOX→anti-DR5 is presumably mediated by reduced protein levels of the IAPs survivin and XIAP. These two IAPs seem to be bound and neutralized by SMAC/Diablo and HTRA2/Omi, respectively, which might explain the low levels of all these four proteins in the apoptosis array. Besides the IAPs survivin and XIAP, the antiapoptotic mitochondrial protein Bcl-2 is also downregulated by DOX→anti-DR5. The down-regulation of these three antiapoptotic proteins is known to sensitize tumor cells to enhanced apoptosis through the extrinsic pathway [39,40]. Interestingly, cytochrome c levels of anti-DR5 + DOX treated cells are significantly higher than the levels in DOX→anti-DR5-treated cells. This might be contributed to the exhaustion of cytochrome c in the cells due to the fast and tremendous apoptosis induction from 1-fold to 54-fold within 6 hours after time-shifted anti-DR5 treatment. Chandra et al. have already described decreased cytochrome c levels in late apoptotic cells [41]. Bad and Bid acting as proapoptotic proteins appeared to be downregulated by anti-DR5 + DOX and DOX→anti-DR5 as shown by both the Apoptosis Array and Western blot, respectively. However, the most potent inducers of apoptosis are the caspase-cleaved, truncated forms of these proteins, while full-length protein levels are decreased [42,43]. A truncated, approximately 10-kDa form of Bid was detected in Western blot analysis, which was increased in anti-DR5 + DOX and DOX→anti-DR5-treated cells. The existence of high amounts of truncated Bid in anti-DR5 + DOX and DOX→anti-DR5 treated cells underlines that cleavage of Bid is critical and prognostic for enhanced apoptosis induction through cross-linking of intrinsic and extrinsic apoptosis pathways [44].

Taken together, the split Luc apoptosis reporter in combination with fluorescence measurement allowed us to accelerate the optimization of dosage, application, and combination schedules in a GBM xenograft model. By monitoring apoptosis in vivo, we demonstrated that the sequential application of DOX followed by anti-DR5 synergistically enhances apoptosis that can already be detected 6 hours after anti-DR5 treatment. We showed that DOX-induced recruitment of DR5 to the cell surface and the proliferation inhibitory effect of DOX result in a higher susceptibility of D54-caspase-3/7 GloSensor tumor cells to anti-DR5 treatment and, consequently, to the superiority of the sequential application of DOX before anti-DR5. The result of our studies may provide useful information regarding clinical study protocols and, thus, may support the clinical development of successful combination therapies with anti-DR5 antibodies.

Acknowledgments

The authors thank Peter Bruenker and Erwin van Puijenbroek for providing anti-DR5 antibody, Michaela Hook for creating Figure 2, and Franz Osl for assisting in conducting animal experiments. T.G.W. thanks Michael Stürzl for supervising his PhD thesis. T.P. thanks the Roche Postdoc Fellowship (RPF) Program for support.

Abbreviations

5-FU

5-fluorouracil

A750

Alexa Fluor 750 (fluorescence dye)

Bid

BH-3 interacting domain death agonist

DEVD

aspartate-glutamate-valine-aspartate cleavage sequence for caspases-3/7

DOX

doxorubicin

DR5

death receptor 5

FACS

fluorescence-activated cell sorting

GBM

glioblastoma multiforme

Luc

luciferase

SCID

severe combined immunodeficient

TRAIL

TNF-related apoptosis-inducing ligand

XIAP

X-linked inhibitor of apoptosis protein

Footnotes

1

A.R. and S.G. were supported by the National Institutes of Health (P01CA085878). T.G.W., T.P., and W.S. are employees of Roche Diagnostics GmbH. S.G. and A.R. have no conflicts of interest.

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