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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Ther. Author manuscript; available in PMC Feb 10, 2010.
Published in final edited form as:
PMCID: PMC2819149
NIHMSID: NIHMS170790

Cyclophosphamide Increases Transgene Expression Mediated by an Oncolytic Adenovirus in Glioma-Bearing Mice Monitored by Bioluminescence Imaging

Abstract

Approaches to improve the oncolytic potency of replication-competent adenoviruses include the insertion of therapeutic transgenes into the viral genome. Little is known about the levels and duration of in vivo transgene expression by cells infected with such “armed” viruses. Using a tumor-selective adenovirus encoding firefly luciferase (AdΔ24CMV-Luc) we investigated these questions in an intracranial mouse model for malignant glioma. Luciferase expression was detected by bioluminescence imaging, and the effect of the immunosuppressive agent cyclophosphamide (CPA) on transgene expression was assessed. Intratumoral AdΔ24CMV-Luc injection led to a localized dose-dependent expression of luciferase. Surprisingly, this expression decreased rapidly during the course of 14 days. In contrast, mice injected with nonreplicating Ad.CMV-Luc demonstrated stable transgene expression. Treatment of mice with CPA in combination with AdΔ24CMV-Luc retarded the loss of transgene expression. Staining of mouse brains for inflammatory cells demonstrated decreased tumor infiltration by immune cells in CPA-treated mice. Moreover, in immunodeficient NOD/SCID mice loss of transgene expression was less rapid and not prevented by CPA treatment. Together, our data demonstrate that transgene expression and viral replication decrease rapidly after intratumoral injection of oncolytic adenovirus in mouse brains and that treatment with the immunomodulator CPA prolongs viral-mediated gene expression.

Keywords: glioma, oncolytic adenovirus, transgene expression, bioluminescence imaging, immune suppression

Introduction

Replication-competent (oncolytic, replication-conditional, replication-compromised) viruses are being investigated as novel anti-cancer agents for various types of cancers [1], including malignant glioma [24]. One feature of such viruses is related to the capacity to engineer therapeutic transgenes into their genome to allow for multimodal therapy. Such “armed” therapeutic viruses (ATVs) allow the action of therapeutic proteins to be combined with the anti-tumor properties of the viral infection. Several oncolytic herpes and adenoviruses that ferry such therapeutic transgenes have been described [510]. However, to date, little is known about the levels and duration of transgene expression in cells infected by such armed viruses.

AdΔ24 is an oncolytic adenovirus that is being widely used as a backbone in the construction of various ATVs [1114]. The tumor specificity of AdΔ24 is based upon a partial deletion in the CR2 domain of the adenoviral E1A gene, thereby abrogating the binding of the E1A protein to pRB [15]. The adenoviral E3 region has been removed in AdΔ24, allowing the insertion of transgene expression cassettes. AdΔ24 and modifications of this backbone have shown therapeutic efficacy in various in vitro and in vivo models of malignant glioma [1418].

In the current study, we investigated the in vivo kinetics of transgene expression by this type of oncolytic adenovirus and strategies to increase transgene levels. We employed an AdΔ24 virus encoding the firefly luciferase marker gene (AdΔ24CMV-Luc) [19]. The levels of luciferase expressed in tumors infected with this virus were studied in mice bearing intracranial glioma xenografts. Luciferase expression was monitored noninvasively using a cooled charge-coupled device camera. A time-dependent decrease in luciferase gene expression in tumors injected with this oncolytic virus in vivo was found. Concomitant administration of the immunomodulating agent cyclophosphamide (CPA) retarded the observed time-dependent decrease in luciferase expression and decreased the inflammatory reaction mediated by the oncolytic virus in the athymic nude mouse strain. This effect was not observed in nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice, which harbor severe defects in both innate and adaptive immunologic function. Our data indicate that immune-mediated factors play a role in the rapid loss of transgene expression and viral replication in mouse brains and that pretreatment with immunomodulating agents can retard this process.

Results

Intratumoral Transgene Expression from an Oncolytic Adenovirus in a Mouse Glioma Model is Transient

We injected athymic nude mice bearing intracranial human gliomas with a dose range (106, 107, or 108 plaque-forming units (pfu)) of the replication-competent oncolytic adenovirus encoding luciferase (AdΔ24CMV-Luc) or with 107 or 108 pfu of a replication-defective adenoviral vector encoding firefly luciferase (Ad.CMV-Luc). We monitored transgene expression noninvasively by bioluminescence imaging up to day 14 post-infection (Fig. 1). Further follow-up was not possible due to mice becoming symptomatic from tumor growth. The replication competence of the virus leads one to expect a time-dependent increase in luciferase activity as a function of increased viral titers by ongoing replicative cycles. However, despite evidence of ongoing viral replication at all three dosages of AdΔ24CMV-Luc as detected by hexon staining on cryosections at time of sacrifice (not shown), we observed a rapid decrease in intracranial luciferase expression. On the other hand, luciferase expression from both dosages of the replication-defective Ad.CMV-Luc remained high throughout the course of the experiment. Quantitative analysis of luciferase signals demonstrated that by day 14 luciferase expression had decreased 23.2-, 21.3-, and 29.4-fold relative to the first measurements taken at 24 h postinfection for the 106, 107, and 108 dose, respectively (Fig. 2). These results thus show that transgene expression mediated by an oncolytic adenovirus in human gliomas in mice is transient and limited in time.

FIG. 1
In vivo imaging of luciferase activity in mice bearing U-87ΔEGFR intracranial tumors treated with an intratumoral injection of a dose range of AdΔ24CMV-Luc (106, 107, 108 pfu) or Ad.CMV-Luc (107 or 108 pfu). Individual mice are shown with ...
FIG. 2
Quantitative image analysis of intensity of light emitted from intracranial tumor sites demonstrates loss of transgene expression in mice treated with replication-competent adenovirus and endurance of transgene expression in mice treated with replication-deficient ...

Intratumoral Virus Replication Rapidly Decreases with Time

To visualize the kinetics of viral replication and spread in the intracranial tumors, we injected nude mice bearing U-87ΔEGFR xenografts with 5×107 pfu AdΔ24CMV-Luc and sacrificed them on day 3, 5, or 7 postinfection and at the time of manifestation of symptoms (day 16). Analysis of viral replication by immunohistochemical staining for adenovirus hexon proteins demonstrated that virus replication was distributed in a patchy manner through- out the tumor. There were a large number of uninfected tumor cells in relation to the cells that were infected. A rapid decrease in hexon-positive areas of the tumor was seen as the tumor continued to grow over time (Fig. 3). These findings are in accordance with the loss of transgene expression from a replicating adenovirus as detected by bioluminescence imaging.

FIG. 3
Immunohistochemical staining for adenovirus hexon proteins in cryosections of brains of U-87ΔEGFR tumor-bearing mice treated with 5×107 pfu AdΔ24CMV-Luc and harvested at day 3, 5, 7, and 16 after virus injection. Tumor borders ...

Cyclophosphamide Retards Loss of Transgene Expression in the Athymic Mouse Glioma Model

We have previously reported that cyclophosphamide limits the effects of the host response against an oncolytic HSV1 in human and rat gliomas in rats, allowing prolonged viral replication and transgene expression to take place [20,21]. We thus sought to determine if the addition of CPA enhanced luciferase activity in AdΔ24CMV-Luc-infected human gliomas in mice. To this end, we injected intracranial U-87ΔEGFR-bearing nude mice with 5×107 pfu AdΔ24CMV-Luc with or without CPA pretreatment. We chose this viral dose based on the observation that mice are not cured at this dose and hence tumor load would not be a limitation for the replication of the virus, as demonstrated in Fig. 3. Fig. 4 depicts the color-coded images of the individual mice at days 4, 9, and 12 post-virus injection. Control mice (left column) again demonstrate a gradual loss of transgene expression with time in tumors infected with AdΔ24CMV-Luc. However, CPA treatment (right column) appeared to retard the decrease in luciferase activity over time. In fact, image analysis revealed that CPA allowed for a relatively small decline (6-fold) in luciferase activity in tumors compared to untreated tumors (33-fold), and by the 14th day there was an order of magnitude difference in tumors treated with CPA+virus compared to PBS+virus (Fig. 5). Significant differences in total luciferase expressed between these groups were detected on days 5, 9, 12, and 15 (P <0.05).

FIG. 4
In vivo imaging of luciferase activity in mice bearing U-87ΔEGFR intracranial tumors treated with intratumoral injection of AdΔ24CMV-Luc alone (left column) or in combination with CPA treatment (right column). Shown are the individual ...
FIG. 5
Quantitative image analysis of intensity of light emitted from intracranial tumor sites demonstrates prolonged high-level transgene expression in mice treated with CPA + AdΔ24CMV-Luc (CPA) compared to PBS +AdΔ24CMV-Luc (Control). Shown ...

The Activated Metabolite of CPA Does Not Affect Transgene Expression Mediated by AdΔ24CMV-Luc in Vitro

One potential mechanism that could explain the in vivo CPA effect might relate to the cytotoxic mode of action of the drug. In fact, adenoviral oncolysis has been reported to be enhanced in combination with various cytotoxic agents [2226]. If such a mechanism were operative, one would expect to see an increase in viral-mediated transgene expression in vitro, too. To assess whether the observed in vivo enhancement of luciferase expression resulted from direct effects of CPA on transgene expression by the AdΔ24CMV-Luc-infected tumor cells, we also treated human U-87ΔEGFR cells with this combination in vitro. We incubated cells with a concentration of the activated metabolite of CPA (4-hydroperoxy-CPA; 4-HO2-CPA) that was not toxic to tumor cells and subsequently infected them with m.o.i. 1, 4, and 16 of AdΔ24CMV-Luc. On days 4 and 7 postinfection, we analyzed the cells for viability and luciferase expression. Treatment with a nontherapeutic dose of 4-OH-CPA (3 μM) had no significant effect on the viability of glioma cells at day 4 or 7 (m.o.i. 0). Whereas the oncolytic activity of AdΔ24CMV-Luc was not detectable at day 4, a dose-dependent cytotoxic effect of the virus was evident by day 7. This effect was not significantly altered by 3 μM 4-OH-CPA (Fig. 6A). This dose of 4-OH-CPA also did not significantly alter luciferase expression at day 4 or 7 (Fig. 6B). As expected, when we administered higher doses of 4-OH-CPA, we found an enhanced cell killing effect in combination with the virus, although transgene expression was not significantly altered (data not shown). These results indicate that the activated metabolite of CPA does not affect transgene expression levels by direct action on tumor cells.

FIG. 6
(A) Viability of U-87ΔEGFR glioma monolayers after treatment with a dose range of AdΔ24CMV-Luc alone (●) or in combination with a nontoxic dose of 4-HO2-CPA (■) was determined using the WST-1 assay on days 4 and 7 postinfection. ...

Decreases in Inflammatory Cell Infiltrates in Gliomas Treated with CPA and Oncolytic Virus

Since the effect of CPA did not appear to be related to the drug’s direct action on the cell, we sought to determine if effects on inflammatory infiltrates were present. It is important to note that athymic nude mice still produce small numbers of T cells, as well as still being able to produce interferons, natural killer cells, and macrophages [27]. These residual components of the immune system can contribute to an inflammatory response to oncolytic adenoviruses. We harvested mouse brains 72 h and 5 days after AdΔ24CMV-Luc infection and at the time of occurrence of symptoms due to tumor growth (days 16–21). Analysis of tumors of animals treated with PBS + AdΔ24CMV-Luc demonstrated the presence of numerous CD45- and CD68-positive cells at 72 h post-infection, 20.9±3.3 and 17.2±3.7%, respectively (Fig. 7A, top). CD45 is expressed on cells of hematopoietic lineage, especially lymphocytes, and to a lesser extent on microglial cells [28]. CD68 is expressed at high levels on monocytic cells, macrophages, and microglial cells. Adenoviral hexon immunopositivity was detected in patches throughout the tumor. When we administered CPA in combination with AdΔ24CMV-Luc (Fig. 7A, bottom) CD45 and especially CD68 immunopositivity was decreased to14.2±5.6 and 4.4±1.9%, respectively, while hexon immunopositivity remained similar at this time point. At day 5 postinfection CD45 and CD68 immunopositivity in tumors of animals treated with CPA + AdΔ24CMV-Luc, 16.1 ±4.5 and 11.1 ±2.6%, respectively, was still decreased (Fig. 7B, bottom), compared to 26.2±1.2 and 22.8±2.1% positive cells, respectively, in the PBS+AdΔ24CMV-Luc tumors (Fig. 7B, top). Moreover, at this time point, adenoviral hexon protein immunopositivity was quantitatively and qualitatively increased in CPA-treated animals compared to the PBS controls. This increase correlated with enhanced bioluminescence signals in CPA-treated mice compared to controls starting at this time point. Analysis of tumors harvested at time of sacrifice (days 16–21) revealed no detectable differences in CD68 or CD45 immunopositivity and only very few hexon-positive cells within each tumor (not shown). These results provided suggestive evidence that CPA induced a transient inhibition of inflammatory cell infiltration in tumors treated with the oncolytic AdΔ24CMV-Luc and that this inhibition is associated with an increase in adenovirus replication.

FIG. 7
Immunohistochemical staining for common leukocyte antigen (CD45), monocytic cells/macrophages (CD68), and adenoviral hexon proteins in intracranial U-87ΔEGFR tumors of athymic mice treated with AdΔ24CMV-Luc+PBS (top) or AdΔ24CMV-Luc+CPA ...

Cyclophosphamide Does Not Prevent Loss of Transgene Expression in the NOD/SCID Mouse Glioma Model

To verify that an immune response and not other physiological components are being affected by the CPA treatment in the nude mouse glioma model, we repeated this experiment in the NOD/SCID strain. These mice are characterized by a functional deficit in T, B, and NK cells; absence of circulating complement; and defective antigen-presenting cells [29]. We monitored transgene expression noninvasively by bioluminescence imaging up to day 9 after AdΔ24CMV-Luc injection. Further follow-up was not possible due to mice becoming symptomatic from tumor growth, which occurred more rapidly than in the nude mice. Data analysis of acquired images revealed a relatively small but significant decline (fivefold, P <0.05) in luciferase activity in controls by day 4 postinfection, which then stabilized up to day 9. Importantly, CPA treatment did not prolong high-level transgene expression in these mice (Fig. 8A). Immunohistochemical analysis of cryosections of the brains of these animals harvested on day 9 postinfection demonstrated the presence of CD45- and CD68-positive cells in PBS+ virus-treated tumors, 9.3 ±3.4 and 10.9 ±1.1%, respectively, albeit in lower amounts and smaller in size than we detected in virus-treated tumors of athymic mice. In addition, the staining pattern for adenovirus hexon protein was more pronounced and widespread than seen in athymic mice on day 7. CPA treatment of NOD/SCID mice did not affect CD45, CD68, and hexon staining patterns in virus-treated tumors (Fig. 8B).

FIG. 8
(A) Quantitative image analysis of intensity of light emitted from intracranial tumor sites in NOD/SCID mice showing transgene expression levels of mice treated with CPA+ AdΔ24CMV-Luc (CPA) or PBS+AdΔ24CMV-Luc (Control). Shown are mean ...

Discussion

The use of ATVs is being exploited as a novel therapeutic modality for tumors. One assumption in this therapy has been that viral-mediated transgene expression would increase as long as tumor burden persists due to the recurring cycles of viral replication with viral progeny production and subsequent infection and replication in additional tumor cells. Indeed, in vitro studies in glioma cells have shown a 3 log higher level of luciferase expression 48 h after infection with replication-competent adenovirus encoding luciferase compared to replication-deficient control vectors [30]. Moreover, in primary glioma spheroids a ninefold increase in luciferase expression was found during the course of 9 days using replication-competent adenovirus, compared to a fourfold decrease using replication-deficient adenovirus encoding luciferase [31]. However, the level and duration of in vivo transgene expression from an oncolytic adenovirus have not been monitored thus far.

Using bioluminescence imaging, we assessed the pattern of transgene expression from an oncolytic adenovirus delivered to a tumor. Others have reported the utility of bioluminescence imaging for monitoring transgene expression from implanted luciferase-expressing (tumor) cells [32,33] or after systemic or intratumoral administration of luciferase-encoding nonreplicating (targeted) viral vectors or naked plasmid DNA [3436]. Moreover, viral infections such as encephalomyelitis by Sindbis virus [37] and spread to various anatomic sites of herpes simplex virus type 1 [38] can be detected using bioluminescence imaging. Furthermore, Yu et al. have employed this system to detect tumors and metastases by injection of “tumor-seeking” bacteria or vaccinia virus encoding luciferase [39]. Thus far, bioluminescence imaging has not been applied for direct in vivo visualization and monitoring of transgene expression from an oncolytic virus.

We demonstrate here that intratumoral injection of the firefly luciferase-encoding tumor-selective adenovirus AdΔ24CMV-Luc leads to dose-dependent expression of luciferase at the tumor site. This expression can be detected for at least 15 days. However, we also demonstrate that transgene expression actually decreases fairly precipitously upon intratumoral injection of this oncolytic virus. This decrease was unexpected and distinct from the luciferase expression pattern after infection with the nonreplicating vector Ad.CMV-Luc, which remained high throughout the course of the experiment. The decrease in transgene expression from AdΔ24CMV-Luc may result from less efficient viral spreading and reinfection within the tumor than was predicted by in vitro studies [30,31]. This is supported by studies on wild-type adenovirus spreading in subcutaneous xenografts [40,41]. In addition, immune-mediated factors also appear to play a role in this phenomenon.

It has been reported that in the immunoprivileged environment of the brain, nonreplicating adenoviral vectors provide stable and prolonged gene expression [42,43] by comparison to peripheral organs [44]. However, above a certain threshold, acute vector-mediated toxicity and chronic inflammation lead to a decline in transduction persistence [45]. These results are congruent with the stable luciferase expression in brains of mice injected with 107 or 108 pfu of the replication-defective Ad.CMV-Luc. The decline in luciferase expression after intratumoral injection of the replication-competent AdΔ24CMV-Luc in athymic mice is associated with a CD45+ and CD68+ cellular infiltrate, suggestive of an inflammatory reaction. Bioluminescence imaging of mice treated with a combination of AdΔ24CMV-Luc and the immunosuppressive agent cyclophosphamide demonstrated that CPA can retard the loss of transgene expression, resulting in prolonged high-level luciferase expression at the tumor site. Immunohistochemical analysis of tumor sections confirmed the bioluminescence data by detecting the presence of ongoing viral replication and reduction of immune cell infiltrate in CPA-treated mice.

Further support for the hypothesis that an inflammatory response contributes to the inhibition of oncolytic adenovirus-mediated transgene expression and replication in intracranial tumors was obtained using NOD/SCID mice. The results from this experiment demonstrate that in a background of multiple defects in innate and adaptive immune function, transgene expression from an oncolytic adenovirus declines much less rapidly. Moreover, treatment with the immunomodulating agent CPA has no significant effect on levels of transgene expression.

The finding that in vivo transgene expression mediated by an oncolytic adenovirus decreases after tumor injection is in agreement with our previous findings [46,47] with an oncolytic herpes simplex virus (HSV). CPA’s ability to retard the loss of transgene expression mediated by an adenovirus also confirms the previous findings with an oncolytic HSV. In addition, for the first time, we also show that CPA’s ability to do this is not dependent on species since all our previous studies were reported in rats, while the current experiments were performed in mice. The mechanism of CPA action in providing for improved transgene expression does not depend on a direct effect on cellular metabolism since it does not occur in vitro. Its effect thus must involve an effect on the host or on the tumor stroma. We observed a rapid upregulation of cells expressing CD45 and CD68 in virally treated tumors of athymic mice. These cells represent lymphocytic and monocytic/microglial populations, which have been shown to be involved in inflammatory reactions against replication-defective adenoviral vectors as well [48]. Interestingly, a phase I clinical trial of an oncolytic adenovirus in humans with glioma also showed a lymphocytoid/plasma cell infiltrate in injected tumors [3]. Taken in conjunction with our data, this is suggestive evidence of a rapid host response to the oncolytic virus infection.

The immunosuppressive action of CPA may thus provide an avenue to circumvent the host response and enhance viral oncolysis and the therapeutic effects of transgenes delivered by oncolytic viral agents. However, the fact that CPA can only partially rescue the decrease in luciferase signal suggests that either the effect of CPA on the immune response is partial or other factors also contribute to the rapid decrease in transgene expression from the oncolytic adenovirus. Such factors may include ineffective viral dissemination within the tumor caused by murine stroma or tumor hypoxia [40,41]. The design of oncolytic viruses or combination therapies that overcome these barriers, together with concomitant immune suppression, is expected to lead to more effective oncolytic viral therapies.

Materials and Methods

Cell culture

The Ad5 E1-transformed human embryonal kidney cell line 293, the human lung carcinoma cell line A549, and the glioma cell line U-87MG were purchased from the ATCC (Manassas, VA, USA). U-87ΔEGFR was a generous gift from Dr. H. J. Su Huang (University of California at San Diego, La Jolla, CA, USA). All cells were cultured in DMEM supplemented with 10% FBS and antibiotics (Life Technologies, Paisley, UK).

Recombinant adenoviruses

A recombinant E1-deleted, replication-deficient adenovirus expressing the cytomegalovirus (CMV) immediate early promoter-driven firefly luciferase, Ad.CMV-Luc, was provided by Dr. R. D. Gerard (University of Texas Southwestern Medical Center, Dallas, TX, USA). Construction of the conditionally replicative adenovirus with an expression cassette for firefly luciferase in the E3 region, AdΔ24CMV-Luc, has been described previously [19]. Viruses were plaque purified, propagated on 293 cells (Ad.CMV-Luc) or on A549 cells (AdΔ24CMV-Luc), and purified by CsCl gradient according to standard techniques. The E1Δ24 mutation and/or CMV-Luc insertion was confirmed by PCR on the final products. Particle titer was determined by absorbance measurements at 260 nm and the functional titer (pfu) was determined by end-point dilution titration on 293 cells according to standard techniques. The particle/pfu ratio was 11 for AdΔ24CMV-Luc and 12.5 for Ad.CMV-Luc.

In vitro assays

Glioma cells were plated subconfluently at 2.5×103 cells per well in 96-well plates. After 24 h culture medium was replaced by medium containing a dose range of 4-HO2-CPA (kindly provided by Dr. S. M. Ludeman at Duke University, Durham, NC, USA). In solution, 4-HO2-CPA is spontaneously converted to 4-hydroxy-CPA, which is the first active metabolite formed in the metabolism of CPA. After another 24 h cells were infected in triplicate with a dose range of AdΔ24CMV-Luc. Viability of cells was assessed by WST-1 assay (Roche Diagnostics, Mannheim, Germany) at days 4 and 7 postinfection. Results are presented as percentage of uninfected controls. In parallel plates, luciferase expression in cell extracts was measured using the luciferase chemiluminescence assay system (Promega Benelux, Leiden, The Netherlands) on days 4 and 7 after infection. After the culture medium was removed, lysis buffer was added to the wells and the whole plate was snap-frozen on dry ice. After the samples thawed, the luciferase activity in the cell lysates was measured using a Lumat LB 9507 spectrofluorometer (EG&G Berthold, Bad Wildbad, Germany).

In vivo tumor model

Intracranial glioma xenografts were established in adult female athymic mice (CD1 nude; Charles River, Wilmington, MA, USA) or NOD/SCID mice (NOD.SCID/NCr; NIH–National Cancer Institute, Frederick, MD, USA) by stereotactic injection of 105 U-87ΔEGFR cells in 3 μl Hanks’ buffer into the right frontal lobe, 2.5 mm lateral to the bregma, at a depth of 3 mm. Injections were performed under anesthesia by intraperitoneal injection of 2 mg ketamine and 0.4 mg xylazine in 0.9% saline.

In the first experiment mice were inoculated stereotactically into the same coordinates as the tumor cells on day 5 post-tumor cell injection with a dose range of 106, 107, or 108 pfu AdΔ24CMV-Luc or with 107 or 108pfu replication-defective Ad.CMV-Luc in 3 μl PBS. Two mice per virus dose were monitored by bioluminescence imaging three times per week up to day 14. Animals were monitored daily and sacrificed upon appearance of symptoms evident for moribund decline due to intra-cerebral tumor growth, such as paralysis and lethargy. For analysis of viral spreading, an additional four animals were injected with 5×107 pfu AdΔ24CMV-Luc and sacrificed on day 3, 5, or 7 or at the time of appearance of symptoms. The brains of these animals were snap-frozen for sectioning and immunohistochemical analysis.

For the second experiment, nude mice were randomly divided into two groups after receiving stereotactic tumor cell injection. On day 3, a single intraperitoneal injection of either PBS or 200 mg/kg CPA (Cytoxan; Mead Johnson) diluted in PBS was administered. On day 5 animals received 5×107 pfu AdΔ24CMV-Luc in 3 μl PBS inoculated stereotactically into the same coordinates as the tumor cells. Two animals from each group were sacrificed at 72 h and at 5 days postinfection. Brains were removed and frozen for (immuno)histological analysis. Bioluminescence imaging was performed on the remaining four animals every other day up to day 16. Mice were monitored daily and sacrificed upon appearance of symptoms evident of moribund decline due to intracerebral tumor growth, such as paralysis and lethargy. Brains were removed and frozen for (immuno)histological analysis.

For the third experiment in NOD/SCID mice, the same protocol for tumor cells, virus, and CPA injection as in the second experiment was applied. Bioluminescence imaging was performed on the PBS- (n =6) and CPA- (n =7) treated mice on days 1, 2, 4, and 9 after AdΔ24CMV-Luc injection. Mice were monitored daily and sacrificed on day 9 after imaging due to the appearance of symptoms evident of intracerebral tumor growth. Brains were removed and frozen for (immuno)histological analysis.

Bioluminescence imaging

Imaging of AdΔ24CMV-Luc-induced luciferase expression in athymic mice was performed with a cryogenically cooled high-efficiency CCD camera system (Roper Scientific, Duluth, GA, USA) custom developed at MGH using protocols similar to those described previously [49,50]. Mice were injected intraperitoneally with 4.5 mg D-luciferin substrate (Molecular Imaging Products, Ann Arbor, MI, USA) and images were acquired 15 min after D-luciferin administration. A surface image of each animal was acquired by dim polychromatic illumination. The luciferase activity within the brain of each animal was then measured by recording photon counts in the CCD with no illumination. Imaging of NOD/SCID mice was performed similarly using the Night OWL LB981 bioluminescence imaging system (Berthold Technologies, TN, USA).

After data acquisition, postprocessing and visualization were performed with a home-written program (CMIR-Image) with integrated image display and analysis suite. Regions of interest were defined and the sum and standard deviation of the photon counts within regions of interest were then calculated. For visualization purposes, the bioluminescence images were fused with the corresponding white light surface images as a transparent pseudocolor overlay. Statistical analysis between virus and virus+CPA groups at each time point was conducted with the two-tailed Student t test.

Immunohistochemistry

Immunohistochemical staining for adenovirus hexon proteins and mouse macrophages and leukocytes was performed on acetone-fixed cryosections obtained from brains of U-87ΔEGFR tumor-bearing mice. Briefly, immunohistochemical staining was performed for adenovirus using the goat antiadenoviral hexon protein antibody 1056 (Chemicon Europe, Hampshire, UK), for monocytes/macrophages using the rat anti-mouse CD68 antibody (Serotec, Oxford, UK), and for hematopoietic cells using the rat anti-mouse CD45 antibody to leukocyte common antigen (ITK, Uithoorn, The Netherlands). Negative controls were assessed by omitting the primary antibody. Bound primary antibodies were detected using HRP-conjugated rabbit anti-goat or rabbit anti-rat antibodies and exposure to the chromogen diaminobenzidine (DakoCytomation, Glostrup, Denmark), followed by counterstaining with Mayer’s hematoxylin. Quantification of CD45 and CD68 immunopositivity was performed in four representative areas of the tumor using ImageJ software (NIH, http://rsb.info.nih.gov/ij).

Statistical analysis

Data from the first in vivo and in vitro experiments are presented as means+SD. Data from the second and third in vivo experiments are shown±SEM. Statistical analysis between groups was conducted with the two-tailed Student t test.

Acknowledgments

The authors thank Ida van der Meulen-Muileman for technical assistance and Ted Graves for assistance in quantitative analysis of imaging data. This work was supported by the Dutch Cancer Society, Grant VU2002-2594; The Ter Meulen Fund of the Royal Netherlands Academy of Arts and Sciences; the VUMC Institute for Cancer and Immunology to M.L.M.L.; and NIH Grants P01 CA69246, NS41571, and R01 CA85139 to E.A.C. In vivo imaging experiments were supported in part by R24 CA92782 and P50 CA86355 to R.W.

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