© The Author 2006. Published by Oxford University Press.
ARTICLE |
Dynamic Monitoring of Oncolytic Adenovirus In Vivo by Genetic Capsid Labeling
Affiliations of authors: Division of Human Gene Therapy (LPL, HNL, IPD, JGD, TG, SY, DTC, MY), Departments of Medicine, Pathology, Surgery, and Obstetrics and Gynecology and the Gene Therapy Center (IPD, DTC, MY), University of Alabama at Birmingham, Birmingham, AL
Correspondence to: David T. Curiel, MD, PhD, Division of Human Gene Therapy, 901 19th St. S., BMR2-502, Birmingham, AL 35294-2172 (e-mail: curiel{at}uab.edu) or Masato Yamamoto, MD, PhD, Division of Human Gene Therapy, 901 19th St. S., BMR2-410 Birmingham, AL 35294-2172 (e-mail: masatoy{at}uab.edu).
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ABSTRACT |
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Background: Conditionally replicative adenoviruses represent a promising strategy to address the limited efficacy and safety issues associated with conventional cancer treatment. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus replication and spread and host–vector interactions in vivo have not been completely evaluated. Methods: We developed a noninvasive dynamic monitoring system to detect adenovirus replication. We constructed capsid-labeled E1/E3-deleted and wild-type adenoviruses (Ad-wt) by fusing the minor capsid protein IX with red fluorescent proteins mRFP1 and tdimer2(12), resulting in Ad-IX-mRFP1, Ad-IX-tdimer2(12), and Ad-wt-IX-mRFP1. Virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to primary receptor (coxsackie adenovirus receptor) were analyzed using real-time quantitative polymerase chain reaction, cell viability (MTS) assay, and fluorescence microscopy. Athymic mice (n = 4) carrying xenograft tumors that were derived from A549 lung adenocarcinoma cells were intratumorally inoculated with Ad-wt-IX-mRFP1, and adenovirus replication was dynamically monitored with a fluorescence noninvasive imaging system. Correlations between fluorescence signal intensity and viral DNA synthesis and replication were calculated using Pearson's correlation coefficient (r). Results: The red fluorescence label had little effect on viral DNA replication, encapsidation, cytopathic effect, thermostability, and coxsackie adenovirus receptor binding. The fluorescent signal correlated with viral DNA synthesis and infectious progeny production both in vitro and in vivo (in A549 cells, r = .99 and r = .65; in tumors, r = .93 and r = .92, respectively). The replication efficiency of Ad-wt-IX-mRFP1 in vivo was variable, and replication and viral spreading and persistence were limited, consistent with clinical observations. Conclusions: Genetic capsid labeling provides a promising approach for the dynamic assessment of oncolytic adenovirus function in vivo.
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INTRODUCTION |
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Cancer is the second leading cause of disease-related mortality in humans after heart disease despite technologic advances in clinical management (1). Conventional tumor therapies, including surgery, radiotherapy, and chemotherapy, often have poor efficacy and may have undesirable side effects (2–4). Oncolytic viral treatment (also known as virotherapy) has been proposed as a promising alternative to surgery, radiotherapy, and chemotherapy. Conditionally replicative adenoviruses represent a candidate agent in this endeavor and have the potential for transductional and transcriptional targeting (5–8), and their rapid evaluation in clinical trials has demonstrated their safety. However, to date, conditionally replicative adenoviruses, when used as single agents, have not displayed the anticipated efficacy for cancer therapy (9). Oncolytic adenovirus function in humans therefore needs to be carefully explored. Current clinical trial protocols and methods cannot provide the interval endpoint data necessary to fully study fundamental issues such as the extent of replication and spread, specificity, viral persistence, and host–vector interactions. The lack of tools to directly and dynamically observe the performance of conditionally replicative adenoviruses in vivo has been a major impediment in realizing the clinical utility of replicative adenoviral agents.
Current methods of vector detection include chemical labeling, DNA and RNA quantification or hybridization, immunohistochemistry, and reporter gene expression. Although these methods have been operative for certain in vitro and in situ studies of adenovirus biology and gene therapy, their limitations are evident when they are used in replicative vector systems. Most of these terminal assays only allow examination of a particular sample and of one moment in time. However, the adenovirus oncolytic mechanism revolves around the concept that the initial virus amplifies and spreads to eventually yield a tumorwide therapeutic effect (9–11). Such dynamics cannot be captured and represented by static analysis. The shortcomings of current vector detection methods are further complicated by the need to acquire multiple biopsies using an invasive procedure that is prone to sampling error and is concomitantly impractical for repeated monitoring of the entire tumor (12–15). Reporter genes can only provide indirect and relative information with respect to virus replication and localization based on transgene expression.
The ideal approach for evaluating the replication and dissemination of oncolytic adenoviral agents should directly measure the viral mass that accrues from the initial administration without compromising replication capacity and be capable of noninvasive detection. To this end, we hypothesized that the detection of viral capsid proteins genetically fused with an imaging reporter would provide such an index of viral replication and localization. Previously, we established an adenovirus capsid labeling strategy by fusing the minor capsid protein IX with enhanced green fluorescent protein (EGFP) (16). Herein, we expand the capsid labeling technology to develop red fluorescent protein (RFP)–labeled adenoviruses with conserved viral function to monitor adenoviral replication in vivo.
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METHODS |
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Cell Culture
Human embryonic kidney 293 (American Type Culture Collection [ATCC], Manassas, VA), human embryonic retinoblast 911 (17), human lung adenocarcinoma A549 (ATCC), BALB/c mouse transformed liver BNL-1NG-A.2 (ATCC), and Chinese hamster ovary (CHO) (ATCC) cells were maintained according to the suppliers' protocols. The cells were incubated at 37 °C and 5% CO2 under humidified conditions.
Recombinant Adenovirus Construction
All viruses were constructed by homologous recombination in Escherichia coli (18). All parental plasmids have been previously described: pShuttle-cytomegalovirus (CMV) (AdEasy system; Qbiogene, Irvine, CA), pRSETB-mRFP1 and pRSETB-tdimer2(12) (19), pSh1pIXNheI (20), and pShuttle-wt-IX-EGFP (21). Shuttle plasmids for the E1/E3-deleted (replication-deficient) vectors were constructed using restriction cloning as follows: pShuttle-E1-CMV-mRFP1 
pShuttle-CMV/BglII/HindIII + pRSETB-mRFP1/BamHI/HindIII; pShuttle-E1-CMV-tdimer2(12) pShuttle-CMV/BglII/HindIII + pRSETB-tdimer2(12)/BamHI/HindIII; pShuttle-IX-mRFP1 pShlpIXNhe/BmtI/blunt + pRSETB-mRFP1/BamHI/EcoRI/blunt; pShuttle-IX-tdimer2(12) pSh1pIXNhe/BmtI/blunt + pRSETB-tdimer2(12)/BamHI/EcoRI. All blunted fragments were generated with large Klenow fragment (New England Biolabs, Beverly, MA). pShuttle-wt-IX-mRFP1 was made by ligating pShuttle-IX-mRFP1/BspHI/MfeI with the BspHI/MfeI fragment from pShuttle-wt-IX-EGFP containing the wild-type E1 region; pShuttle-wt-IX-tdimer2(12) was similarly constructed. All E1/E3-deleted final genomes were made by recombining the above shuttle plasmids (linearized with PmeI) with pAdEasyDS, a modified pAdEasy plasmid that allows double-selection recombination (unpublished data). The wild-type shuttle vector Ad-wt-IX-mRFP1, with the red fluorescent protein label IX-mRFP1, was recombined with pTG3602DS, a modified E1-deleted pTG3602 plasmid that also allows double-selection recombination. Clones were verified by digestion with restriction enzymes and 0.8% agarose gel electrophoresis. The viruses generated include E1/E3-deleted control vectors (with wild-type [wt] protein IX [pIX]) adenovirus (Ad)-E1-CMV-mRFP1 and Ad-E1-CMV-tdimer2(12), E1/E3-deleted Ad-IX-mRFP1 and Ad-IX-tdimer2(12) with pIX modifications, and wild-type E1/E3 Ad-wt-IX-mRFP1. We were unable to recover Ad-wt-IX-tdimer2(12). Therefore, Ad-wt-IX-mRFP1 served as a surrogate oncolytic replicative vector for our studies.
Virus Propagation and Purification
Replication-deficient viruses were propagated in E1-complementing 911 retinoblast cells, and Ad-wt-IX-mRFP1 was amplified in A549 lung cancer cells. Viruses were purified by double cesium chloride (CsCl) ultracentrifugation (AdEasy system; QBiogene) and were dialyzed against phosphate-buffered saline (PBS, 1 mM KH2PO4, 0.15 mM NaCL, 5.5 mM Na2HPO4) containing 0.5 mM Mg2+, 0.9 mM Ca2+, and 10% glycerol. Final aliquots of virus were analyzed for viral particle titer (absorbance at 260 nm), transducing unit titer, and cytopathic effect unit titer. Based on a previously described protocol (22), the transducing unit was determined by infecting 911 cells in 96-well plates with 1:10 serial dilutions of the virus and counting the number of red fluorescent cells 2 days after infection (n = 3). The same plate was assayed with an MTS viability assay (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium; Promega, Madison, WI) to determine the viral dilution that is cytotoxic to 50% of the cells. The experiment was performed once in triplicate. Based on the number of cells seeded (15000/well), the cytopathic effect unit was calculated such that 1 unit is defined as the amount of virus that causes cytopathic effect in one 911 cell in 2 days (23). All viruses were stored at –80 °C until use.
Characterization of Virus Gradient Fractions
For the fractionation studies, Ad-E1-CMV-mRFP1, Ad-E1-CMV-tdimer2(12), Ad-IX-mRFP1, and Ad-IX-tdimer2(12) were each propagated in 10 150-mm dishes of 911 cells. Cells were harvested by aspiration, and viruses were purified by double CsCl ultracentrifugation as described above, in which the top and bottom bands were retained in the same sample after two centrifugation steps, yielding one gradient from the 10 dishes. After the second centrifugation, fractions of 2 drops each (approximately 100 µL) were collected through a perforation at the bottom of the tube into a 96-well white opaque plate. Plates with the viral fractions were measured with a microplate fluorometer (Fluostar Optima; BMG Labtechnologies, Durham, NC) using a 560/10 nm excitation filter for all viruses, a 585/10 nm emission filter for Ad-IX-tdimer2(12), and a 605/10 nm emission filter Ad-IX-mRFP1. To determine viral DNA content, a sample (10 µL) of each fraction was diluted in 90 µL of 0.5% sodium dodecyl sulfate in PBS and incubated at room temperature for 10 minutes to release the viral genomes. Absorbance at 260 nm was then measured for each sample (MBA 2000; Perkin Elmer, Shelton, CT).
Tracking of Red Fluorescent Adenovirus Infection
A549 cells (2.5 x 105 cells/well) were seeded in phenol red–free growth medium (Dulbecco's modified Eagle [DME]–5% fetal calf serum [FCS]) in six-well plates containing glass coverslips (one per well). The next day, cells were incubated for 1 hour at 4 °C with (two wells) or without (four wells) recombinant adenovirus serotype 5 fiber knob (24) (1 µg/mL) in 500 µL of phenol red-free DME medium containing 25 mM HEPES buffer. Ad-IX-mRFP1 or Ad-IX-tdimer2(12) (10000 viral particles/cell) were added to the infection solution to a total volume of 1 mL. The viruses were allowed to bind to the cells at 4 °C for 1 hour (cell binding and Ad5 knob block). Two wells with the added viruses were further incubated at 37 °C for 1.5 hours (nuclear trafficking). Cells from binding and nuclear trafficking experiments were washed three times with PBS and then fixed to the coverslips with 3% formalin (Tousimis, Rockville, MD) for 10 minutes. Cells were washed three times with PBS, stained with 1 µg/mL Hoescht 33342 (Molecular Probes, Eugene, OR) for 5 minutes at room temperature to visualize nuclear DNA, washed three times with PBS, mounted in mounting medium (Biomeda, Foster City, CA), and sealed to glass slides.
In Situ Detection of Ad-wt-IX-mRFP1 Biodistribution
All methods were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and performed according to their guidelines. Three C57/BL6 mice (8 weeks of age; Charles River Laboratories, Wilmington, MA) were anesthetized with 2% isoflurane at approximately 0.5 L/min, and 200 µL of PBS containing 1011 Ad-wt-IX-mRFP1 viral particles was injected into the tail vein of each mouse. The mice were killed by carbon dioxide gas euthanasia 20 minutes after virus injection, and their organs (lung, kidney, liver, and spleen) were surgically removed and frozen by flash freezing in liquid nitrogen. The samples were stored at –80 °C until sectioning (Minotome PLUS; Triangle Biomedical Sciences, Durham, NC). Sections (5-µm thick) of the frozen organs were fixed onto glass slides and stained with Hoechst 33342 as described above. Glass coverslips were mounted and sealed as above.
Fluorescence Microscopy
The slides from the cellular tracking and biodistribution experiments were observed under epifluorescence microscopy. This procedure was performed with an inverted IX-70 microscope (Olympus, Melville, NY) equipped with a Magnifire digital charge-coupled device camera (Optronics, Goleta, CA). Images were acquired with a 100x objective using oil immersion and digitally deconvolved with Iris version 4.15a (25) by applying the Richardson-Lucy algorithm with 15 iterations. An image of a single fluorescent virus particle with a high signal-to-noise ratio was used to estimate the point spread function, as suggested by the software documentation. Red fluorescent protein and Hoechst stain images were merged using Adobe Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA).
DNA Packaging Analysis
DNA packaging was analyzed using a previously reported protocol (26). Briefly, 911 cells (1 x 105/well) were infected with the control and pIX-modified vectors at 1 cytopathic effect unit/cell in 24-well plates. On days 1, 2, 3, and 4 after infection, the cells were collected by aspiration, and DNA was extracted using the QIAamp DNA Blood Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Half of the cells were not pretreated before DNA extraction and were used to determine total intracellular viral DNA. The other half was used to measure intracellular encapsidated viral DNA. The cells were incubated in deoxycholate buffer (0.4% sodium deoxycholate, 0.1 M Tris-Cl, pH 9.0, and 20% ethanol) before DNA extraction to avoid disrupting the viral capsid and in 500 mM spermine to remove unencapsidated viral DNA. The viral genome copy number was determined by TaqMan quantitative real-time polymerase chain reaction (PCR) using E4-specific primers (LightCycler System; Roche Applied Science, Indianapolis, IN). The experiment was performed once in triplicate.
Cytopathic Effect Assay
911 cells (n = 5000) were infected with the control and pIX-modified E1/E3-deleted vectors (0.5, 0.05, and 0.005 cytopathic effect unit/cell) in 100 µL of phenol red–free DME–5% FCS (five replicates for each condition and five wells of noninfected cells as negative controls). Cytopathic effect was measured by an MTS assay (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium; Promega) on days 0, 2, 4, 6, 8, and 10 after infection. Results are presented as the percentage of noninfected cells after subtracting the blank values (medium only).
Thermostability Assay
Thermostability was analyzed using a modified version of a previously reported protocol (20). Samples of E1/E3-deleted control and pIX modified vectors (106 transducing units in 80 µL of PBS) were incubated at 45 °C for 0, 5, 10, 20, and 40 minutes. Transducing unit infectious titers were then determined for the samples using the above-mentioned procedure.
TaqMan Real-Time Quantitative PCR Binding Assay
All steps were carried out with 1% bovine serum albumin (BSA)–PBS buffer. Suspended CHO and A549 cells (2 x 105 in 100 µL) in test tubes were incubated with 100 µL of buffer alone or buffer containing 2.5 µg/mL recombinant fiber knob (to examine blocking of the primary adenovirus receptor, CAR) at 4 °C with vigorous shaking for 1 hour. Ad-E1-CMV-mRFP1, Ad-E1-CMV-tdimer2(12), Ad-IX-mRFP1, or Ad-IX-tdimer2(12) were then added (5000 virus particles/cell) to the cells. The virus–cell mixtures were incubated with shaking for another hour at 4 °C. The cells were then washed three times and collected for total DNA preparation using the QIAamp DNA Blood Mini Kit, and the viral genome copy number was determined using TaqMan quantitative real-time PCR with E4 primers, as described above.
In Vitro Correlation of pIX-mRFP1 Signal With Replication
A549 (human lung adenocarcinoma, replication permissive, 5000 cells/well) and BNL-1NG-A.2 (BALB/c transformed liver, 5000 cells/well) cells were infected with 1, 0.1, 0.01, and 0.001 cytopathic effect unit/cell of Ad-wt-IX-mRFP1 and Ad-IX-mRFP1 (E1/E3-deleted) in five white opaque 96-well plates (n = 6 for each condition). Fluorescence was measured daily with a fluorometer (Fluostar Optima, BMG Labtechnologies, Durham, NC). On days 2, 4, 6, 8, and 10 after infection, the cells and medium (<100 µL) were collected from the wells, which were subsequently washed with 100 µL of PBS. The cells, medium, and washes were transferred to the corresponding sample microcentrifuge tubes to give a total volume <200 µL. All samples were subjected to three freeze-thaw cycles in a dry ice bath. The tubes were then centrifuged at 18 188 xg and 4 °C for 10 minutes; 30 µL of the supernatant was reserved to determine the transducing unit titer, and the rest (170 µL) was processed for viral DNA using the QIAamp DNA Blood Mini Kit and quantified by TaqMan real-time quantitative PCR with E4 primers, as described above. The correlation coefficient (Pearson's r) was calculated with the CORREL function in Microsoft Excel, Office 2003 (Microsoft Corp., Redmond, WA).
Fluorescence-Based In Vivo Optical Imaging
pIX-mRFP1 fluorescence was detected noninvasively using a custom-built optical imaging system. Briefly, a cryogenically cooled, back-illuminated Princeton Instruments VersArray:1KB digital charge-coupled device camera (Roper Scientific, Trenton, NJ) with a liquid nitrogen autofill system was mounted on top of a light-tight enclosure. The camera was coupled with a 50-mm Nikkor f/1.2 lens (Nikon, Melville, NY) for image acquisition. Excitation light for fluorescence imaging was delivered by a Dolan-Jenner Fiber-Lite MH-100 metal halide light source equipped with a dual-fiberoptic gooseneck. Excitation and emission filter wheel assemblies were integrated with the light source and lens. Bandpass filters included 490/10 and 560/10 nm for excitation and 535/30 and 605/55 nm for emission (Chroma Technology, Rockingham, VT).
A549 cells (7.5 x 106) were inoculated in the left and right flanks of athymic nude mice (n = 6) (National Cancer Institute-Frederick Animal Production Area, Frederick, MD) to establish tumors. When the tumors reached 5–10 mm in diameter (in approximately 3 weeks), a single intratumoral injection of Ad-wt-IX-mRFP1 (1010 viral particles in 10 µL total volume of PBS) was performed for each tumor without deliberate spreading of the virus with the needle. Mice (up to three) were placed in the imaging chamber and maintained with 2% isoflurane gas anesthesia at a flow rate of approximately 0.5–1 L/min per mouse (Highland Medical Equipment, Temecula, CA). Images were acquired with WinView/32 software (Roper Scientific). To detect red fluorescence, images were captured at f/4 and f/2 with 2- and 5-second exposure times using two filter combinations: 560/605 and 490/605 (excitation/emission). The former filter setting applies to a red fluorescence signal and the latter configuration pertains to background autofluorescence. A bright-field image was also taken at f/16 for 1 second and at the lowest light level. Background subtraction was performed in WinView/32 after scaling the background image with a factor determined from areas surrounding tumors of individual mice (27). The positive signal from background subtracted images was segmented and analyzed in ImageTool 3.0 (The University of Texas Health Science Center in San Antonio, TX) for integrated density (the product of mean intensity and signal area). Index color image overlays were created in Photoshop 7.0 (Adobe) using the segmentation or thresholding parameters determined in ImageTool.
In Vivo Correlation of pIX-mRFP1 Signal With Replication and Dynamic Monitoring
All mice (n = 6) were imaged daily and analyzed using the above procedure. On day 6 after injection, the day after the maximal signal intensity was observed, four mice were killed. Dissected tumors were imaged in their anatomic position ex vivo and then frozen at –80 °C until use. The tumors were homogenized with the Mini-Beadbeater (BioSpect Products, Bartlesville, OK) and incubated with liver digest medium (4 µL/mg of tumor; Invitrogen, Carlsbad, CA) for 1 hour at 37 °C to further disrupt the tissue. Samples of the tumor homogenate (40 µL [or roughly 10 mg] for large tumors and 10 µL [or approximately 2.5 mg] for small tumors) were used for total DNA determination and viral DNA copy number as described above. The transducing unit titer and cytopathic effect unit titers were determined in the homogenate using the same methods described for purified virus. However, in this case, the cytopathic effect unit was determined 10 days after infection and would give higher values than the same assay read 2 days after infection. All results are presented as total values scaled for the entire tumor mass. The correlation coefficient (Pearson's r) was calculated with the CORREL function in Microsoft Excel. Two of the six original mice were maintained and imaged over the 30-day experiment. Images were processed and analyzed accordingly.
Comparison of In Situ Detection of pIX-mRFP1 Signal With Hexon Staining
Established A549 tumors injected with Ad-wt-IX-mRFP1 were excised on day 7 after injection and frozen in a dry ice–ethanol bath. Frozen tumor sections (5 µm) were fixed onto glass slides with 3% formalin, blocked with 1% BSA–PBS, and probed with a polyclonal goat anti-hexon antibody at approximately 20 µg/mL (1 : 200 dilution; Chemicon, Temecula, CA) for 1 hour at room temperature. The slides were then washed with 1% BSA–PBS and incubated with an Alexa Fluor 488–labeled secondary donkey anti-goat antibody (1:200 dilution; Molecular Probes, Eugene, OR). The slides were washed again with 1% BSA–PBS, and the cells were counterstained with Hoechst 33342 (Molecular Probes) and prepared for fluorescence microscopy as described above.
Statistical Analysis
All statistical analyses were performed with a two-sided single-factor analysis of variance test. P values <.05 were considered statistically significant.
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RESULTS |
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Incorporation of pIX-mRFP1 and pIX-tdimer2(12) Into Viral Particles
We initially constructed E1/E3-deleted viruses with carboxyl-terminal fusions of pIX with monomeric and tandem dimer red fluorescent proteins [mRFP1 and tdimer2(12), respectively] (19). After standard CsCl double ultracentrifugation of the two vectors, we observed that the colors of the empty (top) and mature (bottom) viral bands were different from those obtained from purified conventional unlabeled vectors: Ad-IX-mRFP1 was purple and Ad-IX-tdimer2(12) was pink (Fig. 1, A, data not shown). The difference in color between these two vectors is probably due to the excitation and emission properties of the fluorescent proteins (19). Applying our previously established approach (21), we collected fractions of each viral gradient and analyzed each sample for red fluorescence and viral DNA content. Fluorescent peaks were detected for both the bottom and top bands of the two viruses, which coincided with the optical absorbance peaks of viral DNA (Fig. 1, B). Red fluorescent purified viral particles could be easily visualized using fluorescence microscopy for both vectors (Fig. 1, C).
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Tracking of Ad-IX-mRFP1
To examine the use of the red fluorescent adenoviruses for virus tracking, we incubated A549 cells with Ad-IX-mRFP1 at 4 °C for 1 hour to allow virus binding but not internalization. Fluorescence microscopy revealed distinct binding of Ad-IX-mRFP1 particles to the plasma membrane (Fig. 2, A, cell binding). In another experiment, we allowed the viruses to bind to the cells at 4 °C for one hour and then at 37 °C for 1.5 hours. After the incubation at 37 °C, numerous viruses were detected at the nuclear membrane or inside the nucleus (Fig. 2, A, nuclear trafficking). We also examined whether preincubation with recombinant Ad5 knob would mitigate virus binding. Indeed, few particles remained bound to the A549 cells after washing when knob blocking was implemented (Fig. 2, A, Ad5 knob block), suggesting that the pIX-mRFP1 fusion did not negatively affect the virus's interaction with its primary coxsackie adenovirus receptor. Similar results were also obtained for Ad-IX-tdimer2(12) (data not shown).
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Detection of Ad-wt-IX-mRFP1 in Tissue
To test the ability to detect red fluorescent virus in situ, we injected Ad-wt-IX-mRFP1 (1011 virus particles) into the tail veins of C57/BL6 mice. Twenty minutes after injection, the lungs, kidneys, liver, and spleen were surgically removed, frozen, and sectioned for microscopy. In the lungs, red particles were occasionally visualized around blood vessels and endothelial cells. Likewise, very few particles were detected in the kidneys. Numerous viral particles were seen in the liver, with accumulation being especially prominent in the sinusoids, where the Kupffer cells reside. Single particles (orange arrows, Fig. 2, B) were observed in the hepatocytes. This localization pattern in the liver closely resembles what we observed with Ad-wt-IX-EGFP (16). A substantial amount of virus was also detected in the spleen, mostly in the marginal zones between the white and red pulp (Fig. 2, B).
DNA Encapsidation Efficiency of Red Fluorescent Viruses
Our goal was to establish genetic labeling of adenovirus with minimal perturbation of normal viral function to retain efficient oncolytic activity. One important function for viral replication is the DNA encapsidation efficiency of the red fluorescent adenoviruses. To analyze this function, we assayed lysates from 911 cells infected with the two red fluorescent adenoviruses and their respective E1-CMV expression vector controls for both total and encapsidated viral DNA copy number using TaqMan quantitative real-time PCR (Fig. 3, A, left and middle panels). The data revealed no differences in the total viral DNA replication of the two labeled vectors relative to their controls during 4 days of infection, except for total viral DNA comparison between the tdimer2(12) vectors on day 4 [Ad-E1-CMV-tdimer2(12) versus Ad-IX-tdimer2(12) at day 4, means = 5.50 x 106 versus 9.84 x 106, difference = 4.33 x 106, 95% confidence interval = 2.98 x 106 to 5.69 x 106]. Similarly, there were no differences in encapsidated viral DNA between the two labeled vectors and their controls during the first 3 days of infection. When the data were expressed as encapsidated viral DNA fraction, no differences were noted between the two labeled vectors and their controls (Fig. 3, A, right panel).
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Cytopathic Effect of Red Fluorescent Adenoviruses
The red fluorescent adenoviruses were also evaluated with respect to their ability to induce a cytopathic effect in infected cells. This assay gauges viral function on a more comprehensive level than the previous assays because the cytopathic effect is dependent not only on efficient transduction and virus replication but also on spread to neighboring cells. Any defect in the infection process would result in decreased cytotoxicity. 911 cells were infected with 0.5, 0.05, or 0.005 cytopathic effect units/cell of Ad-E1-CMV-mRFP1, Ad-IX-mRFP1, Ad-E1-CMV-tdimer2(12), and Ad-IX-tdimer2(12). Cell viability was measured every 2 days after infection for a total of 10 days. Under the various conditions examined, there were statistically significant differences noted at some time points and similarities at other time points between the labeled vectors and their controls (Fig. 3). Overall, however, the differences were minor. Moreover, all four viruses achieved total oncolysis with similar kinetics in a dose-dependent manner (Fig. 3, B).
Thermostability of Red Fluorescent Adenoviruses
Adenovirus protein IX functions as a minor protein in capsid stabilization (28,29). To test whether the fusion of red fluorescent proteins to pIX destabilizes the adenovirus capsid structure, we incubated the labeled viruses and their controls at 45 °C. After exposing the viruses to 45 °C for various time intervals, we determined the transducing unit to assess the presence of the remaining infectious virions that survived the temperature stress treatment. No statistically significant decrease in thermostability was detected for Ad-IX-mRFP1 and Ad-IX-tdimer2(12) compared with their controls Ad-E1-CMV-mRFP1 and Ad-E1-CMV-tdimer2(12) after 5 or 10 minutes at 45 °C (Fig. 4, A). After 20 minutes of heat treatment, no infectious viruses remained for any of the four vectors (Fig. 4, A).
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Coxsackie Adenovirus Receptor–Dependent Binding of Red Fluorescent Adenoviruses
We next evaluated the extent of the red fluorescent adenovirus interaction with the Ad5 primary coxsackie adenovirus receptor. Coxsackie adenovirus receptor–deficient CHO cells were chosen as the negative control and high coxsackie adenovirus receptor–expressing A549 cells as the positive control. The viruses were incubated with A549 and CHO cells at 4 °C using vigorous agitation for 1 hour and were then washed to remove unbound viruses. TaqMan real-time quantitative PCR was used to quantify the number of bound virions. As expected, CHO cells showed minimal binding of all four vectors, similar to cell samples with no virus addition (Fig. 4, B). A549 cells, however, demonstrated abundant binding of Ad-IX-mRFP1 and Ad-IX-tdimer2(12) that was equal to that of the control viruses Ad-E1-CMV-mRFP1 and Ad-E1-CMV-tdimer2(12). Similar to the tracking assay, binding was greatly attenuated when the A549 cells were initially incubated with recombinant Ad5 knob (Fig. 4, B).
Correlation of pIX-mRFP1 Signal With DNA Replication and Progeny Production In Vitro
To determine whether the fluorescence intensity of the label itself corresponds to the level of virus mass and replication, we constructed a pIX-mRFP1–labeled virus with intact E1 and E3 regions to serve as a surrogate oncolytic agent. In contrast, a wild-type pIX-tdimer2(12) virus could not be recovered, probably owing to compromise of the pIX function required in packaging full-length genomes (30). As a result, we used Ad-wt-IX-mRFP1 to further investigate the genetic labeling system. We infected A549 and BNL-1NG-A.2 cells in vitro with various amounts of Ad-wt-IX-mRFP1. A549 cells are human lung adenocarcinoma cells that are frequently used to efficiently propagate replication-competent Ad vectors (possessing E1). BNL-1NG-A.2 cells are transformed BALB/c liver cells. Because human adenoviruses in general do not replicate productively in murine cells (31), these two cell lines represent distinct substrates, the former being replication permissive and the latter being replication nonpermissive for human Ad5.
Cell infection was monitored daily over 10 days by measuring the kinetics of red fluorescence resulting from Ad-wt-IX-mRFP1 replication. In BNL-1NG-A.2 cells, no increase in pIX-mRFP1 red fluorescence was detected relative to baseline levels under any of the conditions tested (Fig. 5, A, left panel). By contrast, strong, dose-dependent augmentation of red fluorescence signal with time was observed in A549 cells (Fig. 5, A, middle panel). These data support the concept that the pIX-mRFP1 signal can serve as an index of virus replication. Fluorescence microscopy of BNL-1NG-A.2 and A549 cells infected with Ad-wt-IX-mRFP1 corresponded to the quantitative fluorescence observations (data not shown). A red fluorescence signal was correlated with two parameters of virus replication over the 10-day infection depicted for 0.1 cytopathic effect unit/cell of Ad-wt-IX-mRFP1: viral DNA synthesis (r = .99) and transducing unit titer (r = .65). The poor correlation between fluorescence and transducing unit titer was much improved during the first 8 days, when cellular and medium conditions were suitable for active replication and progeny stability (r = .92) (Fig. 5, A, right panel).
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Correlation of pIX-mRFP1 Signal With DNA Replication and Progeny Production In Vivo
The ultimate utility of our genetic capsid labeling system requires a correlation between fluorescence and viral mass in vivo. To establish such a correlation, we used a fluorescence based noninvasive optical imaging system to detect the replication of Ad-wt-IX-mRFP1 in vivo. Six athymic nude mice with established A549 tumors in the left and right flanks were injected intratumorally with a single dose of Ad-wt-IX-mRFP1 and imaged daily for red fluorescence. After 6 days, tumors from four of the mice were excised, imaged ex vivo, and homogenized. A portion of each tumor homogenate was used to measure viral DNA content, and the clarified supernatant of the remaining homogenate was used to quantify transducing unit titer and cytopathic effect unit titer.
An array of replication patterns in the different tumors were observed, despite the initial similarity in tumor sizes and viral treatment (Fig. 5, B). The variation in fluorescence intensity among the eight tumors allowed us to perform a correlative analysis of the underlying level of viral replication for a wide range of signals. Based on our hypothesis, we expected lower levels of adenovirus in tumors with weaker fluorescence and vice versa. The fluorescence-integrated densities (product of the mean intensity and segmented signal area) were strongly correlated with total viral genome copy number, transducing unit titer, and cytopathic effect unit titer in the tumors (Fig. 5, C, r = .93, r = .92, and r = .97, respectively). Likewise, associating the integrated densities observed in the ex vivo images with these same parameters of adenovirus detectionresulted in even stronger correlation (Fig. 6, A and B, r = .96, r = .97, and r = .97, respectively). We also conducted the same experiment with BNL-1NG-A.2 xenograft tumors (human Ad5 replication nonpermissive) as a negative control, which did not produce any red fluorescence signal (data not shown). In addition to strong correlation between fluorescence signal and viral replication, pIX-mRFP1 localization corresponded with the localization of hexon immunostaining (Fig. 7), a technique that is often used to detect adenovirus in participants of clinical trials (32,33).
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Dynamic Monitoring of Red Fluorescent Ad-wt-IX-mRFP1 Replication In Vivo
We used the genetic adenovirus labeling system to dynamically monitor the replication and oncolysis of Ad-wt-IX-mRFP1 in vivo in two of the six mice with established A549 tumors on the left and right flanks for 30 days. The images shown are for one representative mouse with a strong response to the virus over 30 days (Fig. 6). Initially, different virus behavior between the left and right tumors could be discerned, similar to that observed in the in vivo correlation experiment and other studies (data not shown). Similar to other mice, a peak in pIX-mRFP1 signal intensity was always detected several days following injection, after which the fluorescence appeared to decay over time. For example, the signal peaked at day 2 in the left tumor, whereas the same event occurred at day 4 in the right tumor (Fig. 8, A). We typically observed maximal signal between 2 and 6 days after injection (data not shown). After the signal peaked, it eventually disappeared completely (day 9 for the left tumor and day 20 for the right tumor; Fig. 8, A). Note that the occurrence of a second strong signal in the right tumor starting on day 6 appears to be close to the injection site where granulation tissue eventually formed. Interestingly, the right tumor with the most intense pIX-mRFP1 signal regressed almost completely (day 20); however, it eventually relapsed after the pIX-mRFP1 signal abated, indicating little or no residual viral replication activity after 2 weeks to produce an ongoing antitumor effect. Quantification of the fluorescence signal intensity in the tumors of this mouse further highlights the transient behavior of Ad-wt-IX-mRFP1 replication in vivo (Fig. 8, B). These data show the feasibility of using this genetic capsid labeling system to dynamically monitor adenovirus replication in vivo and to capture the kinetic changes in this process.
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DISCUSSION |
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TopNotesAbstractIntroductionMethodsResultsDiscussionReferences |
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We have created a genetic capsid adenovirus labeling system by fusing the minor capsid protein IX with mRFP1 and tdimer2(12). The capsid fusion protein label was incorporated into virions to allow vector detection in tracking assays and in various tissues with high resolution. Modification of the pIX capsid protein with the red fluorescent proteins had a minimal effect on virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to its primary receptor coxsackie adenovirus receptor. pIX-mRFP1 signal represented the underlying level of adenovirus replication both in vitro and in vivo and correlated well with viral DNA synthesis and infectious progeny production. Furthermore, the localization of pIX-mRFP1 matched results obtained with the conventional hexon staining method used to detect the presence of adenovirus. We also successfully applied the genetic capsid labeling system to dynamically follow the kinetics of adenovirus replication in vivo.
The data we obtained in vivo demonstrate that this new imaging method to detect adenovirus replication greatly differs from conventional vector detection methods. With our method, the kinetics of viral mass and localization could be visualized in real time with a noninvasive procedure. Although our study was designed primarily to validate the basic utility of our system, we observed a number of important findings pertaining to adenovirus oncolytic function. In particular, we found that adenovirus replication was highly variable from tumor to tumor, even in the same mouse. If this differential response is an inherent aspect of intratumoral injection techniques, then studies relying on methods such as tumor size measurements to gauge oncolysis function should be carefully revisited. The natural heterogeneity of tumor microarchitecture and host response to vectors can substantially impact the function of replicative vectors in vivo (34). Therefore, a well-designed conditionally replicative adenovirus evaluation should embody tools that not only gauge tumor response to vectors but also simultaneously delineate vector function. Robust tumor regression should be attributed to oncolytic effect as a consequence of strong viral replication and spread. Indeed, our detection system showed that strong virus replication does lead to regression of the affected tumor (Fig. 8). Furthermore, we noted that weak replication or subsequent attenuation of replication allows the tumor to actively progress (Fig. 8). Without a noninvasive detection method, one that allows repeated monitoring of virus replication, failure or success in achieving a therapeutic effect would be difficult to determine.
What was not clearly ascertained with our adenovirus monitoring system was true spread of virus replication in the tumor, even though we attempted to use a minimal volume (10 µL) of injected virus and deliberately avoided moving the needle to distribute the virus in effect to create an initial small locus of infection from which virus spread could be properly visualized. We have injected a similar amount and volume of virus (1010 virus particles, 10 µL) in mice with larger tumors (greater than 10 mm in diameter), and imaging also did not reveal the degree of spread expected from a replicative adenovirus (data not shown). A positive pIX-mRFP1 signal was not detected in the margins of the tumors, and the intense signal in the center of the tumor never spread far beyond the initial injection site. Our results are consistent with preclinical studies demonstrating the limited ability of oncolytic adenoviruses to spread intratumorally (35), probably owing to confinement by surrounding necrotic and connective tissues (36). The poor lateralization capability of adenovirus in tumors is also suggested by in situ viral DNA hybridization data from clinical trials that show a few focal patches of positive cells rather than gross, widespread presence of viral DNA (12,13,15). For this very reason, most preclinical conditionally replicative adenovirus studies rely on much larger volumes for virus injection (50 or even 100 µL), apply multiple viral administrations, and practice intentional distribution of the virus to achieve widespread infection for an effective antitumor response (37,38). Future studies should be devised to address this issue of virus lateralization and perhaps incorporate strategies to enhance dissemination of progeny virions. Our genetic capsid labeling system would provide the means to evaluate spreading of oncolytic adenoviruses in this respect.
The transient nature of virus replication detected in vivo in this study also raises questions regarding the persistence of adenovirus replication in subcutaneous tumors of athymic nude mice. We have routinely noticed this trend; i.e., a number of mice besides the ones used in this study showed attenuation of pIX-mRFP1 signal in a matter of weeks (data not shown). The short-lived intratumoral replication of adenovirus observed in our experiments corresponds with persistence data obtained in clinical trials. In patients, a peak in circulating viral DNA was typically detected several days after virus administration, indicative of active replication. Yet, over the course of a few weeks, a substantial decrease to baseline followed, indicating clearance of the oncolytic agent (14,39,40). Replicative adenovirus persistence in the tumor requires efficient infection of viable cells for replication as well as effective release and spread to neighboring cells for subsequent infection. Moreover, the virus has to elude the challenge mounted by the host immune system. Our adenovirus monitoring system offers the potential to study these host–vector interactions that are consequential for replicative adenovirus function. It is conceivable that in vivo clearance of the vector may have been due to immunogenicity of not only the native viral capsid proteins but also the red fluorescent protein used to label the exterior of the capsid. Further investigation is needed to determine the extent to which the exposed fluorescent label on the viral capsid contributes to the immune response directed against the vector.
Certain limitations are associated with the use of fluorescence imaging in our genetic capsid labeling system. The detection depth associated with current fluorescence-based optical imaging technology remains limited (41). Additionally, the possibility of achieving tomographic data from fluorescence imaging for volumetric quantification is still under development and not widely available (42). As a result, conventional fluorescence imaging is presently limited to application for superficial or accessible tumors and would not be adequate for accurate quantification of volumetric fluorescence signals. Other imaging ligands that may be more practical for deeper detection within tissue, such as luciferases (43,44) and herpes simplex virus thymidine kinase (45,46), should be considered for genetic capsid labeling of adenovirus.
In summary, we have devised a genetic capsid labeling strategy that allows dynamic monitoring of oncolytic adenoviruses. This in vivo imaging system provides the means to study adenovirus replication, spread, persistence, and antitumor function for the purpose of addressing key issues that are fundamental to the design of replicative adenoviral agents for cancer therapy. Furthermore, capsid-labeled viruses will also have utility for vector targeting and adenovirus biology studies.
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NOTES |
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We thank Dr. Roger Y. Tsien (University of California at San Diego) for providing the mRFP1 and tdimer2(12) constructs. This work was supported with grants from the National Institutes of Health (R01CA083821, R01CA94084, R01DK063615, and R01CA111569), Department of Defense (W81XWH-04-1-0025, W81XWH-05-1-0035, and DAMD17-03-1-0104), and the Medical Scientist Training Program of the University of Alabama at Birmingham.
Funding to pay the Open Access publication charges for this article was provided by the above grant resources.
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Manuscript received May 25, 2005; revised December 2, 2005; accepted December 15, 2005.
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