Vol. 10, 61– 67, January 1, 2004
Clinical Cancer Research
Featured Article
Conditionally Replicative Adenovirus with Tropism Expanded
towards Integrins Inhibits Osteosarcoma Tumor Growth
in Vitro and in Vivo
Adhiambo M. Witlox,1
Victor W. van Beusechem,2 Bonnie Molenaar,2
Hans Bras,3 Gerard R. Schaap,4
Ramon Alemany,5,6 David T. Curiel,2,6
Herbert M. Pinedo,2 Paul I. J. M. Wuisman,1 and
Winald R. Gerritsen2
Department of Orthopedic Surgery, VU University Medical Center,
Divison of Gene Therapy, Department of Medical Oncology, VU
University Medical Center, and Departments of 3Pathology and
4
Orthopedic Surgery, Academic Medical Center, Amsterdam, The
Netherlands; 5Gene Therapy Unit, Institute Catala d’Oncologia,
Barcelona, Spain; and 6Gene Therapy Center, University of Alabama
at Birmingham, Birmingham, Alabama
adenovirus infection of primary OS cells by two orders of
magnitude. Ad5-⌬24RGD was shown to be highly active in
killing human OS cell lines, as well as primary cell cultures.
Furthermore, intratumoral injections with Ad5-⌬24RGD
into established human OS xenografts that were derived
directly from a patient with OS refractory for chemotherapeutic treatment caused a significant tumor-growth delay.
Furthermore, adenoviral particles could be detected in tumor cells 25 days posttumor injection.
Conclusions: Targeting adenovirus toward integrins
rendered OS cells more sensitive to killing by Ad5-⌬24RGD.
These findings suggest that Ad5-⌬24RGD has potential for
use in OS treatment.
Abstract
Introduction
Purpose: The clinical course of osteosarcoma (OS) demands the development of new therapeutic options. Conditionally replicative adenoviruses (CRAds) represent promising agents for the treatment of solid tumors, because
CRAds have an intrinsic replication capacity that allows in
situ amplification and extensive tumor infection through
lateral spread. The CRAd Ad⌬24 has been developed to
replicate selectively in cells with a defective retinoblastoma
(Rb) pathway. Because genetic alterations in the Rb pathway are frequently observed in OS, Ad⌬24 might be useful
in the treatment of this cancer.
Experimental Design: Because the lack of Coxsackie
adenovirus receptor on OS cells limits the efficacy of CRAd
treatment, we explored alternative target molecules on OS.
Insertion of an Arg-Gly-Asp (RGD-4C) integrin-targeting
motif into the adenovirus fiber knob expanded tropism toward the ␣␯␤3 and ␣␯␤5 integrins. The oncolytic capacity of
the CRAd Ad5-⌬24RGD was tested on primary OS cells in
vitro and in vivo.
Results: The ␣␯␤3 and ␣␯␤5 integrins are abundantly
expressed on OS cells. RGD-mediated infection augmented
Osteosarcoma (OS) is one of the most common primary
tumors of bone. It is highly malignant and typically affects
children and young adults (1). Despite improvements in the
treatment of OS, there are still too many patients who cannot
benefit from current treatment modalities (2, 3). The overall
survival with an aggressive chemotherapy regimen before and
after surgery now varies between 50% and 65% (4 – 6). In
addition, current chemotherapeutic agents have a broad spectrum of side effects (7). Therefore, new therapeutic options,
such as gene therapy, are warranted. In the field of adenoviral
vector (Adv)-based gene therapies for OS, several approaches
have been explored, such as suicide gene therapy (8), tumor
suppressor gene therapy (9), and cytokine-based gene therapy
(10). Results of these studies have not yet been translated into
clinical trials. Important limitations in this regard are the failure
of nonreplicating Adv to achieve sufficient tumor-cell transduction and effective solid-tumor penetration.
The development of conditionally replicative adenoviruses
(CRAds) has addressed these limitations. CRAds represent
promising agents for the treatment of solid tumors, because
CRAds have intrinsic replication capacity that allows in situ
amplification and extensive tumor infection through lateral
spread (11). CRAds have been designed following two different
strategies, i.e., by controlling expression of essential early adenovirus genes with tumor-specific promoters (e.g., see Refs. 12,
13) and by deleting viral genes encoding proteins that are
necessary to complete the viral lytic cycle in normal cells, but
not in cancer cells (14 –16).
A critical step determining CRAd efficacy is the infection
efficiency dependent on expression of the primary adenovirus
receptor, the Coxsackie adenovirus receptor (CAR; Ref. 17).
Low CAR expression on primary-tumor cells has been described to limit the efficacy of adenovirus-based therapy for
several types of cancers, including brain, bladder, and pancreatic
cancer (18 –21). Recently, we demonstrated that Adv-infection
efficiency is also compromised in OS, because of low or absent
1
2
Received 4/14/03; revised 8/4/03; accepted 8/25/03.
Grant support: This study is supported by The Netherlands Organization of Scientific Research (NWO-AGIKO 920-03-167). Dr. van
Beusechem is supported by a fellowship of the Royal Netherlands
Academy of Arts and Sciences (KNAW). Dr. Curiel is supported by
National Cancer Institute Grant Canine Crad R01, R01 CA93796.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Winald R. Gerritsen, VU University Medical
Center, Medical Oncology, Division Gene Therapy, De Boelenlaan
1117, 1081 HV Amsterdam, The Netherlands. Phone: 31-20-4449622;
Fax: 31-204448168; E-mail: [email protected].
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CRAd Inhibits Osteosarcoma Tumor Growth
CAR expression (22). Therefore, other, more effective routes of
infection for OS should be explored. Previously, it has been
shown that incorporation of a cyclic Arg-Gly-Asp (RGD-4C)
sequence that interacts with ␣␯ integrins into the adenovirus
fiber-knob circumvents low CAR expression by providing an
alternative viral entry pathway (23). In addition, recent studies
have shown that a CRAd encoding retinoblastoma protein binding-deficient E1A proteins and the RGD-4C-fiber, Ad5⌬24RGD (24), exhibited oncolytic potency on CAR deficient
cancer cells (25, 21). Therefore, we hypothesized that Ad5⌬24RGD could also be effective in OS tumors.
In this study, we first explored the expression of integrins
on OS cell lines and primary OS cells. Next, we tested the effect
of the RGD-4C insertion in the fiber knob of a nonreplicative
Adv on OS transduction. Finally, we assessed the efficacy of
Ad5-⌬24RGD on a broad panel of primary OS cell cultures in
vitro and on primary OS s.c. xenografts in vivo.
Materials and Methods
Cells and Culture Conditions. MG-63 (Ref. 26; courtesy of Dr. C. Löwik, Leiden University Medical Center, The
Netherlands), MNNG-HOS (American Type Culture Collection,
Manassas, VA; Ref. 27), SaOs-2 (Ref. 28; courtesy of Dr. F. van
Valen, Westfalische Wilhelms-Universitat Münster, Germany),
and CAL-72 (Ref. 29; courtesy of Dr. J. Gioanni, laboratoire de
Cancerologie, Faculte de Medicine, Nice, France) OS cell lines
were maintained in DMEM supplemented with 10% FCS, 50
IU/ml penicillin, 50 ␮g/ml streptomycin, and 2 mM L-glutamine
(all from Life technologies B.V., Breda, The Netherlands) at
37°C in a 5% CO2 humidified atmosphere.
Patient Material. Fresh tumor material from patients
suspected of having a classic high-grade OS was processed
directly after open biopsy surgery and kept under sterile conditions at 4°C. The biopsies were washed in sterile PBS, cut into
small pieces and digested with Liver Digestion Medium (Life
technologies B.V.) during four consecutive 30-min incubations
in a water bath at 37°C. After each round of digestion, the cells
were collected and washed. Finally, all cells were pooled and
washed. Cells were brought into culture and used for experiments at passage 0 –5. Confirmation of OS tumor cell morphology of all short-term cultures was performed by histopathology.
Patient characteristics have been described previously (22).
Fluorescence-Activated Cell Sorter Analysis for Integrin Expression. OS cells were incubated for 1 h on ice with
anti-integrin ␣v␤3 and ␣v␤5 mouse monoclonal antibodies
(␣v␤3 MAB 1962 and ␣v␤5 MAB 1976; CHEMICON International Inc., Temecula, CA), 1:100 diluted in PBS containing
0.1% BSA, followed by FITC-conjugated rabbit-antimouse IgG
antibody 1:100 (DAKO, Glostrüp, Denmark) for 1 h on ice. The
samples were fixed in PBS/1% formaldehyde and analyzed for
integrin expression on a FACscan (Becton Dickinson) using
Cell Quest software within 24 h after fixation. Integrin expression was considered positive when the relative median fluorescence intensity of integrin stained cells over control cells
was ⬎2.
Recombinant Adenoviruses. A recombinant E1-deleted
Adv expressing the luciferase reporter gene under the control of
the cytomegalovirus promoter, AdCMVLuc, was provided by
Dr. R. D. Gerard (University of Texas Southwestern Medical
Center, Dallas, TX). A similar Adv, containing an integrin
targeting RGD-4C peptide within the fiber HI-loop, AdLucRGD, has been described previously (23). The CRAd
Ad⌬24 (30) was made by homologous recombination in
“E1-complementing 293” cells between the pXC1 (Microbix
Biosystems, Toronto, Canada) derivative pXC1-⌬24, carrying a 24-bp deletion in the pRb-binding CR2 domain in E1A
(15), and pBHG11 (Microbix Biosystems). Ad5-⌬24RGD
(31) carries the same ⌬24 mutation, plus the RGD-4C fiber
modification and adenovirus E3 region from pVK503 (23).
Replication-deficient Adv were propagated on 293 cells;
conditionally replicative adenoviruses were propagated on
A549 cells (American Type Culture Collection). Viruses
were purified using cesium chloride gradient banding and
titrated in parallel by end-point-limiting dilution on 293 cells
to determine plaque-forming units (pfu).
Adenovirus Infection Experiments. OS cell lines were
plated in a 24 wells plate at 2.5 ⫻ 104 cells/well. Primary OS
cells were plated similarly or in a 96-well plate at 5 ⫻ 103 or 104
cells/well, depending on the amount of primary OS cells available. To allow adherence, cells were incubated overnight in
DMEM/10% FCS medium. Adenoviruses were diluted in
DMEM/2.5% FCS, and cells were infected in triplicate at the
indicated multiplicity of infection (MOI). One hour after infection, the adenovirus-containing medium was removed and fresh
DMEM/10% FCS medium was added. Cells were cultured at
37°C until analysis of gene transfer by luciferase-activity measurement or of cell viability by WST-1 conversion assay (see
Colorimetric WST-1 Cell Viability Assay).
Luciferase-Activity Measurement. Twenty-four hours
postinfection, cells were assayed for luciferase expression using
the Luciferase Chemiluminescent Assay System (Promega,
Madison, WI). Medium was removed from the cells, and luciferase reporter lysis buffer was added to the wells followed by a
freeze thaw step. Luminescence was measured during 10 s
immediately after initiation of the light reaction in a Luminat LB
9507 luminometer (EG&G Berthold, Bad Wildbad, Germany).
Values were protein normalized (Bio-Rad Protein Assay, BioRad laboratories, Veenendaal, The Netherlands). Results were
expressed as relative light unit(s) (RLU) per microgram of
protein.
Colorimetric WST-1 Cell Viability Assay. Cell viability was quantified using the colorimetric WST-1 conversion
assay, based on the cleavage of the tetrazolium salt WST-1 by
mitochondrial dehydrogenases in viable cells (Roche Diagnostics, Manheim, Germany), 7 or 11 days after infection. The
culture medium was removed and replaced by 100 ␮l of 10%
WST-1 in culture medium. Culturing was continued until the
formation of formazan dye was optimal. The A450 was measured
on a Bio-Rad Model 550 (Hercules, CA) microplate reader.
WST-1 conversion was expressed as a percentage of the conversion by uninfected control cells.
In Vivo Experiment. Female athymic nu/nu mice,
weighing 25–35 g, obtained from Harlan-CPB (Austerlitz, The
Netherlands) were used. Animals were kept under pathogen-free
conditions and fed a standard laboratory diet (Hope Farms,
Woerden, The Netherlands) ad libitum. The experimental protocols adhered to the rules outlined in the Dutch Animal Exper-
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Clinical Cancer Research
Table 1 Fluorescence-activated cell sorter analysis of integrin
expression on osteosarcoma (OS) cell lines and primary OS cells
Relative median expressiona
OS cells
␣v␤3
␣v␤5
MG-63
MNNG-HOS
CAL-72
SaOs-2
OS-1
OS-1a
OS-2
LOS-5
OS-6
OS-6a
3.1
2.4
2.3
2.9
5.2
5.2
5.8
2.6
6.2
2
11.6
12.6
10.5
7.6
2
2
9.2
2.3
5.4
5
a
Relative median fluorescence intensity of integrin stained cells
over control (non-stained) cells. Values ⬎2 were considered positive.
imentation Act (1977) and the published Guidelines on the
Protection of Experimental Animals by the council of the
European Commission (1986). OS-1a (21) tumor pieces (3 ⫻
3x3 mm), derived directly from a patient with OS and propagated s.c. in nude mice, were xenografted under the skin of
nude mice. OS-1a xenografts maintained gross histological
resemblance to the primary human tumor. In the patient, this
tumor had been treated according to the COSS96 protocol
and did not show any response to treatment. When the
nodules reached 150 –300 mm3, mice received intratumor
injections on 5 consecutive days with 20 ␮l of control medium (n ⫽ 7) or 1 ⫻ 107 pfu Ad5-⌬24RGD (n ⫽ 6).
Injections into the tumor were performed from a different
angle every day. Tumor size was monitored twice a week
using digital calipers. Volume was calculated from the average of tumor length and width according to the formula
4/3␲r3. The mice were sacrificed when the tumors reached a
size of ⬎1000 mm3. The two-tailed, unequal variance Student’s t test from SPSS (SPSS Inc., Chicago, IL) was used to
compare tumor growth rate (time required to reach 5 times
the initial tumor volume) between the different treatment
groups.
Microscopic Examinations. When the tumor reached a
volume of ⬎1000 mm3, mice were sacrificed and tumors were
dissected. Slides of 4% formalin-fixed and paraffin-embedded
tumor were routinely stained with hematoxilin-eosin and immunohistochemically for adenovirus hexon with 1:1600-diluted
goat anti-adenovirus AB1056 (CHEMICON International Inc.),
followed by antigoat-biotin-streptavidin horseradish peroxidase
(K377, DAKO). An antigen retrieval step with 10 mM Tris/1 mM
EDTA was included. Positive anti-adenovirus stained areas
were dissected from paraffin blocks. The dissected samples
were worked up for electron microscopy by a one-step dehydration/postfixation technique (toluene-OsO4), followed by embedding in epoxy resin (32). Samples were examined for adenovirus particles by electron microscopy at ⫻24,000 and
⫻108,000 magnification.
Results
Targeting Toward ␣v Integrins Enhanced Infection of
OS Cells. Previously, we examined CAR expression on OS
cell lines and primary OS cell cultures (22). All OS cell lines,
except SaOs-2, and all primary cells showed low or even absent
CAR expression. In search of alternative target molecules on OS
that would allow more efficient CRAd infection, we examined
␣v␤3 and ␣v␤5 integrin expression by fluorescence-activated
cell sorter analysis (Table 1). All OS cell lines and primary OS
cell cultures expressed at least one of these integrins. To study
the utility of integrins as targets for adenovirus infection of OS,
we compared the gene-transfer efficiency of an adenovirus
vector with native tropism (AdCMVLuc) to that of a derivative
vector with RGD-4C fibers (AdLucRGD). As shown in Fig. 1,
targeting toward integrins strongly enhanced gene-transfer efficiency on OS cell lines and primary OS cell cultures. In particular, the on-average two orders of magnitude augmented transduction of primary cells by AdLucRGD held promise for the
utility of integrin-targeted CRAds for the treatment of OS.
Oncolytic Effect of Ad⌬24 and Ad5-⌬24RGD in Vitro.
To evaluate the efficacy of the CRAd Ad⌬24 on OS cells, we
infected four OS cell lines at high MOI (Fig. 2A). Ad⌬24
effectively killed all OS cell lines, suggesting that a CRAd with
the ⌬24-mutation could be effective for the treatment of OS.
However, when we infected the same panel of OS cell lines or
OS primary cell cultures with Ad⌬24 at low-MOI, only CARpositive SaOs-2 cells were killed (Fig. 2B). In contrast, integrintargeted Ad5-⌬24RGD was highly active in killing all OS cell
lines and primary cell cultures at low MOI (Fig. 2B). Thus, only
Ad5-⌬24RGD was capable of effectively killing CAR-deficient
primary OS cells.
Inhibition of OS Tumor Growth by Ad5-⌬24RGD
Treatment in Vivo. Finally, we investigated if Ad5-⌬24RGD
was also effective against CAR-deficient primary human OS
tumors in vivo. Nude mice carrying established OS-1a xenografts derived from a chemotherapy-resistant primary tumor
were treated by intratumor injections with a total virus dose of
5 ⫻ 107 pfu Ad5-⌬24RGD. The growth curves of tumors
injected with virus or control medium are shown in Fig. 3. The
median tumor growth rate was calculated for both treatment
groups. Control tumors had a median tumor growth rate of 14 ⫾
4 days, whereas tumors injected with Ad5-⌬24RGD exhibited a
significant growth delay (median tumor growth rate of 25 ⫾ 4
days; P ⬍ 0.05).
Fig. 1 Targeting an adenovirus vector toward integrins on osteosarcoma (OS) cell lines and primary OS cells. OS cell lines MG-63,
MNNG-HOS, CAL 72, and SaOs-2 and five different primary OS cell
cultures were transduced with AdCMVLuc (white bars) or AdCMVLucRGD (black bars) at a multiplicity of infection of 100 plaque-forming
unit/cell. Luciferase expression was measured after 24 h. Data shown
are mean ⫹SD from a representative experiment done in triplicate. RLU,
relative light unit(s).
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CRAd Inhibits Osteosarcoma Tumor Growth
lication throughout treated tumors, mainly in tumor cells at the
border of areas with necrosis (Fig. 4B). Hexon staining was
absent in nontreated tumors (data not shown). Areas with positive anti-adenovirus staining were sampled for electron microscopy, which showed 65-nm dense, rounded adenovirus particle
inclusions in tumor cells (Fig. 4, C and D). Electron microscopy
of nontreated tumors did not show such inclusions (data not
shown). Altogether, these findings showed a presence and replication of Ad5-⌬24RGD in treated tumors for up to 25 days
postinjection.
Discussion
Fig. 2 The oncolytic effect of Ad⌬24 and Ad5-⌬24RGD on osteosarcoma (OS) cell lines and primary OS cells. OS cell lines and a panel of
primary OS cell cultures were mock infected (white bars) or infected
with Ad⌬24 (black bars) or Ad5-⌬24RGD (hatched bars) at a multiplicity of infection of 100 plaque-forming unit(s) (pfu)/cell (A) or at a
multiplicity of infection of 1 pfu/cell for OS cell lines or 0.1 pfu/cell for
primary OS cells (B). Cell viability was determined by WST-1 conversion assay on day 7 (A) or 11 (B) postinfection. Results are depicted as
the percentage viability compared with mock-infected cells. Data shown
are mean ⫹SD from a representative experiment done in triplicate.
Histological analysis of tumors exceeding 1000 mm3
showed classical OS features with osteoid calcified areas. In
tumors retrieved from Ad5-⌬24RGD-treated animals, classic
OS features could still be observed, together with posttherapeutic changes of necrosis within viable tumor fields surrounded by
fibrosis (Fig. 4A). Anti-adenovirus staining revealed viral rep-
The clinical course of OS demands the development of new
therapeutic options. CRAds represent promising agents for the
treatment of solid tumors. However, their efficacy in OS tumors
may be hampered by inefficient cell entry because of a lack of
CAR expression (22). We, thus, hypothesized that the efficacy
of CRAds against OS could be improved by expanding their
tropism toward ␣␯ integrins. This was supported by our findings
described herein that ␣v integrins are expressed on primary OS
cells and that a replication-deficient Adv, which was genetically
modified to expose the RGD-4C peptide in the fiber knob,
exhibited dramatically enhanced gene transfer into OS cell lines
and, more importantly, into primary OS cells.
We chose to investigate the effect of CRAd integrin targeting using Ad⌬24 as a backbone. Ad⌬24 encodes E1A proteins with a deletion of eight amino acids in the pRb-binding
CR2 domain that disables their capacity to release E2F from
preexisting pRb/E2F complexes, thereby conferring selective
replication in cells with already dysfunctional pRb control (13).
It has already been shown that infection with Ad⌬24-type
CRAds effectively kills cancer cells in vitro and inhibits tumor
growth in vivo, and replication is reduced in nonproliferating
normal cells and in cancer cells with restored pRb function (13,
14). Because genetic alterations in the Rb pathway are frequent
in OS (24, 33), Ad⌬24-type CRAds appear to be useful agents
for treatment of this disease. Notably, however, the concept of
integrin targeting is relevant for any kind of CRAd, irrespective
of the genome modification conferring selective replication.
Enhancing the infection efficiency of Ad⌬24 by integrin
targeting translated into increased oncolytic effects. On a broad
panel of primary OS cells infected at low MOI, Ad5-⌬24RGD
Fig. 3 Ad5-⌬24RGD efficacy in vivo. OS-1a xenografts (150 –300 mm3) received injections of
PBS (n ⫽ 7) or 1 ⫻ 107 plaque-forming unit(s)
(pfu) of Ad5-⌬24RGD (n ⫽ 6) for 5 consecutive
days. Individual growth curves are shown for each
tumor. Mice were sacrificed when tumor sizes
exceeded 1000 mm3. Arrows indicate days of PBS
or Ad5-⌬24RGD injection.
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Clinical Cancer Research
Fig. 4 Detection of adenovirus replication in OS-1a tumor
xenografts. At a tumor size of
⬎1000 mm3, mice were sacrificed and tumors were dissected. Tumor sections of an
Ad5-⌬24RGD treated tumor
were stained with hematoxylineosin (A) and with an antiadenovirus hexon antibody (B).
Viable tumor fields are surrounded by fibrosis (fb). Large
areas of necrosis (nc) are surrounded by hexon stained cells
(arrows), indicative of adenovirus replication. Areas stained
positive for hexon were sampled for electron microscopy (C
and D). Adenovirus particle inclusions are indicated by arrows. Original magnifications:
⫻20 (A and B); ⫻24,000 (C);
and ⫻108,000 (D). ECM, extracellular matrix.
showed complete oncolysis, whereas Ad⌬24 induced no detectable cell kill at all. Because Ad⌬24, in contrast to Ad5⌬24RGD, lacks the adenovirus E3 region, differences in oncolytic effect may not only be explained by the expanded tropism
of Ad5-⌬24RGD. However, Ad⌬24 did effectively kill the
CAR-positive OS cell line SaOS-2 at low MOI, suggesting that
the infection enhancement of Ad5-⌬24RGD contributed to its
efficacy on CAR-negative OS cells.
Primary cells derived directly from patient tumor samples
represent an in vitro model closer to the patient compared with
cell lines. Therefore, the observation that Ad5-⌬24RGD showed
complete oncolysis of primary OS cells at an MOI of only 0.1
pfu/cell, whereas Ad⌬24 required 100 pfu/cell for this effect, is
of significant importance. The in vitro efficacy of Ad5-⌬24RGD
was confirmed in a relevant in vivo OS tumor model. Ad5⌬24RGD caused a significant tumor-growth delay of human
primary, chemotherapy-resistant, CAR-deficient OS xenografts
in nude mice. Histological analysis showed that Ad5-⌬24RGD
replicated in OS tumor cells for at least 25 days. However, the
CRAd did not completely eradicate the s.c. xenografts. This can
best be explained by inefficient dispersion of the CRAd through
the tumor mass after intratumoral injection. Barriers within the
established tumor, such as connective tissue and tumor matrix,
may limit the spread of virus (34). In OS, osteoid calcification
could be such a hurdle for viral therapy that needs to be
overcome.
Altogether, our data demonstrate that targeting a CRAd
toward integrins enhances its cytotoxicity on OS dramatically
and warrants further exploration of Ad5-⌬24RGD for its utility
in OS treatment. The main challenge in the treatment of OS,
however, is the eradication of metastases. Patients with OS do
not die from the primary tumor; they die from metastatic disease, typically in the lungs. Notably, we have found that ␣v␤3
and ␣v␤5 integrins are expressed on OS lung metastases (data
not shown). Clearly, intratumoral injection as evaluated in this
study is not a feasible option for treatment of lung metastases.
However, systemic delivery is now being explored with several
viro-therapy agents in clinical trials (35), and, thus, the use of
Ad5-⌬24RGD could perhaps be considered for the treatment
of OS.
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CRAd Inhibits Osteosarcoma Tumor Growth
Acknowledgments
We thank D.C.J. Mastenbroek and M.v.d. Bergh Weerman for their
expert technical assistance, as well as K. Suzuki for providing Ad5⌬24RGD.
References
1. Renard, A. J., Veth, R. P., Schreuder, H. W., Pruszczynski, M.,
Bokkerink, J. P., van Hoesel, Q. G., and van Der Staak, F. J. Osteosarcoma: oncologic and functional results. A single institutional report
covering 22 years. J. Surg. Oncol., 72: 124 –129, 1999.
2. Lindner, N. J., Ramm, O., Hillmann, A., Roedl, R., Gosheger, G.,
Brinkschmidt, C., Juergens, H., and Winkelmann, W. Limb salvage and
outcome of osteosarcoma. The University of Muenster experience. Clin.
Orthop., 83– 89, 1999.
3. Link, M. P., Goorin, A. M., Horowitz, M., Meyer, W. H., Belasco, J.,
Baker, A., Ayala, A., and Shuster, J. Adjuvant chemotherapy of highgrade osteosarcoma of the extremity. Updated results of the MultiInstitutional Osteosarcoma Study. Clin. Orthop., 8 –14, 1991.
4. Bacci, G., Briccoli, A., Ferrari, S., Longhi, A., Mercuri, M., Capanna, R., Donati, D., Lari, S., Forni, C., and DePaolis, M. Neoadjuvant
chemotherapy for osteosarcoma of the extremity. Long-term results of
the Rizzoli’s 4th protocol. Eur. J. Cancer, 37: 2030 –2039, 2001.
5. Bacci, G., Ferrari, S., Longhi, A., Forni, C., Bertoni, F., Fabbri, N.,
Zavatta, M., and Versari, M. Neoadjuvant chemotherapy for high grade
osteosarcoma of the extremities: long-term results for patients treated
according to the Rizzoli IOR/OS-3b protocol. J. Chemother., 13: 93–99,
2001.
6. Longhi, A., Fabbri, N., Donati, D., Capanna, R., Briccoli, A., Biagini, R., Bernini, G., Ferrari, S., Versari, M., and Bacci, G. Neoadjuvant chemotherapy for patients with synchronous multifocal osteosarcoma: results in eleven cases. J. Chemother., 13: 324 –330, 2001.
7. Sluga, M., Windhager, R., Lang, S., Heinzl, H., Bielack, S., and
Kotz, R. Local and systemic control after ablative and limb sparing
surgery in patients with osteosarcoma. Clin. Orthop., 120 –127, 1999.
8. Ko, S. C., Cheon, J., Kao, C., Gotoh, A., Shirakawa, T., Sikes, R. A.,
Karsenty, G., and Chung, L. W. Osteocalcin promoter-based toxic gene
therapy for the treatment of osteosarcoma in experimental models.
Cancer Res., 56: 4614 – 4619, 1996.
9. Xu, H. J., Zhou, Y., Seigne, J., Perng, G. S., Mixon, M., Zhang, C.,
Li, J., Benedict, W. F., and Hu, S. X. Enhanced tumor suppressor gene
therapy via replication-deficient adenovirus vectors expressing an Nterminal truncated retinoblastoma protein. Cancer Res., 56: 2245–2249,
1996.
10. Worth, L. L., Jia, S. F., Zhou, Z., Chen, L., and Kleinerman, E. S.
Intranasal therapy with an adenoviral vector containing the murine
interleukin-12 gene eradicates osteosarcoma lung metastases. Clin. Cancer Res., 6: 3713–3718, 2000.
11. Alemany, R., Balague, C., and Curiel, D. T. Replicative adenoviruses for cancer therapy. Nat. Biotechnol., 18: 723–727, 2000.
12. Rodriguez, R., Schuur, E. R., Lim, H. Y., Henderson, G. A.,
Simons, J. W., and Henderson, D. R. Prostate attenuated replication
competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res., 57:
2559 –2563, 1997.
13. Hallenbeck, P. L., Chang, Y. N., Hay, C., Golightly, D., Stewart, D.,
Lin, J., Phipps, S., and Chiang, Y. L. A novel tumor-specific replicationrestricted adenoviral vector for gene therapy of hepatocellular carcinoma. Hum. Gene Ther., 10: 1721–1733, 1999.
14. Bischoff, J. R., Kirn, D. H., Williams, A., Heise, C., Horn, S.,
Muna, M., Ng, L., Nye, J. A., Sampson-Johannes, A., Fattaey, A., and
McCormick, F. An adenovirus mutant that replicates selectively in
p53-deficient human tumor cells. Science (Wash. DC), 274: 373–376,
1996.
15. Fueyo, J., Gomez-Manzano, C., Alemany, R., Lee, P. S., McDonnell, T. J., Mitlianga, P., Shi, Y. X., Levin, V. A., Yung, W. K., and
Kyritsis, A. P. A mutant oncolytic adenovirus targeting the Rb pathway
produces anti-glioma effect in vivo. Oncogene, 19: 2–12, 2000.
16. Heise, C., Hermiston, T., Johnson, L., Brooks, G., SampsonJohannes, A., Williams, A., Hawkins, L., and Kirn, D. An adenovirus
E1A mutant that demonstrates potent and selective systemic anti-tumoral efficacy. Nat. Med., 6: 1134 –1139, 2000.
17. Douglas, J. T., Kim, M., Sumerel, L. A., Carey, D. E., and Curiel,
D. T. Efficient oncolysis by a replicating adenovirus (ad) in vivo is
critically dependent on tumor expression of primary ad receptors. Cancer Res., 61: 813– 817, 2001.
18. Grill, J., van Beusechem, V. W., Van Der Valk, P., Dirven, C. M.,
Leonhart, A., Pherai, D. S., Haisma, H. J., Pinedo, H. M., Curiel, D. T.,
and Gerritsen, W. R. Combined targeting of adenoviruses to integrins
and epidermal growth factor receptors increases gene transfer into
primary glioma cells and spheroids. Clin. Cancer Res., 7: 641– 650,
2001.
19. Li, Y., Pong, R. C., Bergelson, J. M., Hall, M. C., Sagalowsky, A. I.,
Tseng, C. P., Wang, Z., and Hsieh, J. T. Loss of adenoviral receptor
expression in human bladder cancer cells: a potential impact on the
efficacy of gene therapy. Cancer Res., 59: 325–330, 1999.
20. Wesseling, J. G., Bosma, P. J., Krasnykh, V., Kashentseva, E. A.,
Blackwell, J. L., Reynolds, P. N., Li, H., Parameshwar, M., Vickers,
S. M., Jaffee, E. M., Huibregtse, K., Curiel, D. T., and Dmitriev, I.
Improved gene transfer efficiency to primary and established human
pancreatic carcinoma target cells via epidermal growth factor receptor
and integrin-targeted adenoviral vectors. Gene Ther., 8: 969 –976, 2001.
21. Cripe, T. P., Dunphy, E. J., Holub, A. D., Saini, A., Vasi, N. H.,
Mahller, Y. Y., Collins, M. H., Snyder, J. D., Krasnykh, V., Curiel,
D. T., Wickham, T. J., DeGregori, J., Bergelson, J. M., and Currier,
M. A. Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus
gene transfer and oncolysis for human rhabdomyosarcoma cells. Cancer
Res., 61: 2953–2960, 2001.
22. Witlox, M. A., van Beusechem, V. W., Grill, J., Haisma, H. J.,
Schaap, G., Bras, J., Van Diest, P., De Gast, A., Curiel, D. T., Pinedo,
H. M., Gerritsen, W. R., and Wuisman, P. I. Epidermal growth factor
receptor targeting enhances adenoviral vector based suicide gene therapy of osteosarcoma. J. Gene Med., 4: 510 –516, 2002.
23. Dmitriev, I., Krasnykh, V., Miller, C. R., Wang, M., Kashentseva,
E., Mikheeva, G., Belousova, N., and Curiel, D. T. An adenovirus vector
with genetically modified fibers demonstrates expanded tropism via
utilization of a coxsackievirus and adenovirus receptor-independent cell
entry mechanism. J. Virol., 72: 9706 –9713, 1998.
24. Feugeas, O., Guriec, N., Babin-Boilletot, A., Marcellin, L., Simon,
P., Babin, S., Thyss, A., Hofman, P., Terrier, P., Kalifa, C., BrunatMentigny, M., Patricot, L. M., and Oberling, F. Loss of heterozygosity
of the RB gene is a poor prognostic factor in patients with osteosarcoma.
J. Clin. Oncol., 14: 467– 472, 1996.
25. Lamfers, M. L., Grill, J., Dirven, C. M., van Beusechem, V. W.,
Geoerger, B., Van Den Berg, J., Alemany, R., Fueyo, J., Curiel, D. T.,
Vassal, G., Pinedo, H. M., Vandertop, W. P., and Gerritsen, W. R.
Potential of the conditionally replicative adenovirus Ad5-⌬24RGD in
the treatment of malignant gliomas and its enhanced effect with radiotherapy. Cancer Res., 62: 5736 –5742, 2002.
26. Billiau, A., Edy, V. G., Heremans, H., Van Damme, J., Desmyter,
J., Georgiades, J. A., and De Somer, P. Human interferon: mass production in a newly established cell line, MG-63. Antimicrob. Agents
Chemother., 12: 11–15, 1977.
27. Rhim, J. S., Putman, D. L., Arnstein, P., Huebner, R. J., and
McAllister, R. M. Characterization of human cells transformed in vitro
by N-methyl-N⬘-nitro-N-nitrosoguanidine. Int. J. Cancer, 19: 505–510,
1977.
28. Fogh, J., Wright, W. C., and Loveless, J. D. Absence of HeLa cell
contamination in 169 cell lines derived from human tumors. J. Natl.
Cancer Inst. (Bethesda), 58: 209 –214, 1977.
29. Rochet, N., Dubousset, J., Mazeau, C., Zanghellini, E., Farges,
M. F., de Novion, H. S., Chompret, A., Delpech, B., Cattan, N., Frenay,
M., and Gioanni, J. Establishment, characterisation and partial cytokine
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2004 American Association for Cancer
Research.
Clinical Cancer Research
expression profile of a new human osteosarcoma cell line (CAL 72). Int.
J. Cancer, 82: 282–285, 1999.
30. van Beusechem, V. W., van den Doel, P. B., Grill, J., Pinedo, H. M.,
and Gerritsen, W. R. Conditionally replicative adenovirus expressing
p53 exhibits enhanced oncolytic potency. Cancer Res., 62: 6165– 6171,
2002.
31. Suzuki, K., Fueyo, J., Krasnykh, V., Reynolds, P. N., Curiel, D. T.,
and Alemany, R. A conditionally replicative adenovirus with enhanced
infectivity shows improved oncolytic potency. Clin. Cancer Res., 7:
120 –126, 2001.
32. van den Bergh Weerman, M. A., and Dingemans, K. P. Rapid
deparaffinization for electron microscopy. Ultrastruct. Pathol., 7: 55–57,
1984.
33. Benassi, M. S., Molendini, L., Gamberi, G., Ragazzini, P., Sollazzo,
M. R., Merli, M., Asp, J., Magagnoli, G., Balladelli, A., Bertoni, F., and
Picci, P. Alteration of pRb/p16/cdk4 regulation in human osteosarcoma.
Int. J. Cancer, 84: 489 – 493, 1999.
34. Kuppen, P. J., van der Eb, M. M., Jonges, L. E., Hagenaars, M.,
Hokland, M. E., Nannmark, U., Goldfarb, R. H., Basse, P. H., Fleuren,
G. J., Hoeben, R. C., and van de Velde, C. J. Tumor structure and
extracellular matrix as a possible barrier for therapeutic approaches
using immune cells or adenoviruses in colorectal cancer. Histochem.
Cell Biol., 115: 67–72, 2001.
35. Reid, T., Warren, R., and Kirn, D. Intravascular adenoviral agents
in cancer patients: lessons from clinical trials. Cancer Gene Ther., 9:
979 –986, 2002.
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2004 American Association for Cancer
Research.
67
Conditionally Replicative Adenovirus with Tropism
Expanded towards Integrins Inhibits Osteosarcoma Tumor
Growth in Vitro and in Vivo
Adhiambo M. Witlox, Victor W. van Beusechem, Bonnie Molenaar, et al.
Clin Cancer Res 2004;10:61-67.
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