British Journal of Cancer (2006) 94, 1837 – 1844

& 2006 Cancer Research UK All rights reserved 0007 – 0920/06 $30.00
www.bjcancer.com

Different susceptibility of osteosarcoma cell lines and primary cells

to treatment with oncolytic adenovirus and doxorubicin or
cisplatin





 HCA Graat1, MA Witlox1, FHE Schagen2, GJL Kaspers3, MN Helder1, J Bras4, GR Schaap5, WR Gerritsen2,

 PIJM Wuisman1 and VW van Beusechem*,2
 1
Department of Orthopedic Surgery, VU University Medical Center, PO box 7057, Amsterdam 1007 MB, The Netherlands; 2Division of Gene Therapy,

 Department of Medical Oncology, VU University Medical Center, PO box 7057, Amsterdam 1007 MB, The Netherlands; 3Department of Pediatric

 Hematology/Oncology, VU University Medical Center, PO box 7057, Amsterdam 1007 MB, The Netherlands; 4Department of Pathology, Academic

 Medical Center, PO Box 22660, Amsterdam 1100 DD, The Netherlands; 5Department of Orthopedic Surgery, Academic Medical Center, PO Box

Translational Therapeutics

 22660, Amsterdam 1100 DD, The Netherlands






 Despite improvements in treatment regimens for osteosarcoma (OS) patients, survival rate has not increased over the last two

 decades. New treatment modalities are therefore warranted. Preclinical results with conditionally replicative adenoviruses (CRAds)

 to treat OS are promising. One type of CRAd that was effective against OS cells is Ad5-D24RGD. In other types of cancer, CRAds

 have been shown to interact synergistically with chemotherapeutic agents. Chemotherapy for OS often includes doxorubicin and

 cisplatin. Therefore, we explored combination treatment of OS cell lines and primary OS cell cultures with Ad5-D24RGD and

 doxorubicin or cisplatin. On OS cell lines, combination treatment was additive to synergistic. Surprisingly, however, on seven of eight

 primary OS samples no such combination effects were observed. In contrast, in many cases chemotherapy even inhibited CRAd-

 mediated cell killing. The inhibitory effect of doxorubicin on Ad5-D24RGD in primary OS cells appeared to correlate with slow cell

 growth rate; reduced viral replication and absence of chemotherapy-induced G2 cell cycle arrest. Our results point to the possibility

 that, at least for OS, virotherapy and chemotherapy should best not be performed simultaneously. In general, our work underscores

 the importance of testing new genetic anticancer agents and treatment regimens on primary cancer specimens.

 British Journal of Cancer (2006) 94, 1837 – 1844. doi:10.1038/sj.bjc.6603189 www.bjcancer.com

 Published online 30 May 2006

 & 2006 Cancer Research UK



Keywords: conditionally replicative adenovirus; osteosarcoma; primary cell cultures; chemotherapy; virotherapy; combination effects

Osteosarcoma (OS) is the most common primary bone tumour. tumour cells thereby causing specific tumour cell lysis. Released
Patients are treated with an aggressive chemotherapy regimen virus from lysed cells can subsequently infect neighbouring
before and after surgery. Improvement of chemotherapeutic drug tumour cells, which results in lateral propagation of CRAds.
regimens and surgical techniques resulted in an overall event-free Previously, we demonstrated that the CRAd Ad5-D24RGD (Suzuki
survival rate of approximately 50 – 70% (Link et al, 1986; Bacci et al, et al, 2001) shows antitumour efficacy against primary OS cells in
2001; Bielack et al, 2002). Patients with local recurrence after vitro and in vivo (Witlox et al, 2004).
chemotherapy or overt metastatic disease have a much poorer To assess the value of a new treatment modality, it is useful to
outcome. Recent trials indicate that 5-year survival rate has reached investigate its efficacy in combination with conventional anti-
a plateau, with little or no improvement by conventional treatment cancer agents that constitute standard therapy. This is particularly
modalities (Link et al, 1986; Bacci et al, 2001; Bielack et al, 2002). relevant for clinical development, where patients are usually not
New treatment modalities for OS are therefore warranted. withheld from conventional treatment. Conventional chemother-
Conditionally replicative adenoviruses (CRAds) represent a apy for OS often includes cisplatin and doxorubicin. A synergistic
potential new treatment modality for solid tumours including OS antitumour effect of CRAds with doxorubicin or cisplatin has been
(Alemany et al, 2000; Hemminki et al, 2003). Conditionally reported previously for other types of cancer (Heise et al, 1997;
replicative adenoviruses are developed to selectively replicate in You et al, 2000; Li et al, 2001; Portella et al, 2002) and CRAd
therapy combined with chemotherapy has already shown promis-
ing results in solid tumours in phase I and II clinical trials (Khuri
et al, 2000; Lamont et al, 2000; Reid et al, 2001). Therefore, we
*Correspondence: Dr VW van Beusechem; sought to determine a possible future role of Ad5-D24RGD in
E-mail: VW.vanBeusechem@vumc.nl combination with doxorubicin or cisplatin for OS treatment, by
Revised 27 April 2006; accepted 2 May 2006; published online 30 May testing this combination treatment first on OS cell lines and
2006 subsequently on a panel of short-term cultured primary OS cells.
 Chemo-gene therapy for OS
HCA Graat et al
1838
MATERIALS AND METHODS 550 microplate reader (Hercules, CA, USA). Relative WST-1
conversion in treated cells compared to untreated cells was
Cell lines and primary OS cells calculated after subtraction of WST-1 conversion in the absence of
The OS cell lines MG-63, SaOs-2 and U2OS were kindly provided cells. For OS primary cells, differences in viability after treatment
by Dr C Löwik (Leiden University Medical Center, The Nether- with the best of either single agent compared to the combination
lands), Dr F van Valen (Westfalische Wilhelms-Universitat treatment were analysed using a two-sided Student’s t-test.
Münster, Germany) and Dr S Lens (Dutch Cancer Institute, Combination effects in OS cell lines were assessed by a constant
Amsterdam, The Netherlands), respectively, and were maintained ratio combination design in the range of 0.063 to 4-times the IC50
in F12-supplemented Dulbecco’s Modified Eagle Medium (DMEM- of the individual components. The mean combination index (CI)
F12) with 10% fetal calf serum (FCS) and 50 IU ml
1 penicillin plus was calculated from four data points at effective doses yielding 50,
50 mg ml
1 streptomycin (PS) (Life technologies, Breda, The 75, 90 and 95% reduced viability, using the CalcuSyn program
Netherlands) at 371C in a humidified, 5% carbon dioxide (Biosoft, Ferguson, MO, USA). This program applies the combina-
atmosphere. tion-index equation described by Chou and Talalay (Chou and
Eight primary and fresh OS tumour samples were retrieved from Talalay, 1984). Combination index values below 0.9 were defined
three patients before chemotherapy treatment was started (OS-6, 8 as synergism; between 0.9 and 1.1 as additive; and above 1.1 as
and 16), from one patient after chemotherapy (OS-5A), and from antagonism. For simplicity, mutual exclusivity was assumed in
two patients before (OS-11 and 12) and after (OS-11A and 12A) these combination experiments.
chemotherapy. All patients were treated with chemotherapy
including high dose of cisplatin and doxorubicin. All patients Quantitative PCR for adenoviral genomes
had high-grade OS. Tumour material was processed directly after
Cells were plated at a density of 5  103 cells well
1 in a 96-wells
Translational Therapeutics

biopsy. In brief, tumour pieces were washed in sterile phosphate-

plate and infected with Ad5-D24RGD at a multiplicity of infection
buffered saline (PBS) and minced. Liver digest medium (Life
(MOI) of 50 plaque-forming units (PFU) per cell. After 1 h, a dose
Technologies) was added during four consecutive 30-min incuba-
of doxorubicin at approximately IC10 (drug concentration result-
tions at 371C. Cells were collected, washed and cultured in DMEM-
ing in 10% inhibition in growth) was added. After 31 h, cells were
F12 with 10% FCS, PS and 2.5 mg ml
1 fungizone (Life techno-
harvested and lysed in PBS by three freeze/thaw cycles. Cleared
logies) at 371C in a humidified, 5% carbon dioxide atmosphere.
lysate (100 ml) was treated overnight with 5 mg proteinase K (Sigma,
Osteosarcoma morphology of all primary cell cultures was
St Louis, MO, USA) in a water bath at 371C. Quantitative PCR was
confirmed by histological analysis. Primary cultures were used
performed with a hexon-specific primer set: sense, 50 -ATG ATG
until passage eight.
CCG CAG TGG TCT TA-30 , antisense, 50 -GTC AAA GTA CGT GGA
AGC CAT-30 and the FastStart DNA Master SYBR Green I kit
p53 reporter assay (Roche Diagnostics, Mannheim, Germany) according to the
manufacturer’s protocol. The amount of viral DNA templates
To investigate phenotypic p53 status, primary OS cells were plated was calculated on the basis of a standard curve generated by
at a density of 5  104 cells per well in a 24-wells plate. Cells were amplification of a dilution series of the hexon gene containing
transfected with either the p53-dependent reporter plasmid PG13- plasmid pBHG11 (Microbix biosystems, Toronto, Canada).
Luc, or with the negative control construct MG15-Luc, as described
previously (van Beusechem et al, 2002). Relative luciferase
expression of PG13-Luc compared to MG15-Luc was used to Cell growth assay
determine p53-status. Ratios of 0.5 – 2.0 were considered to To determine the doubling time of OS cell cultures, cells were
represent a p53-deficient status, ratios between 2 and 10 probably plated at a density of 5  104 cells well
1 in a six-wells plate. The
represent a heterogeneous cell population consisting of p53- cells were cultured for up to 7 days and counted on days 1, 3, 5 and
deficient and wild-type cells, and ratios above 10 represent 7 using a Coulter Counter (Beckman Coulter, Fullerton, CA, USA).
functional p53 status. The doubling time was calculated from three independent
experiments and is given as the average with standard deviation
Adenovirus and chemotherapeutic agents (s.d.).

The CRAd Ad5-D24RGD lacks 24 base pairs encoding eight amino Cell cycle analysis
acids in the pRb-binding domain of E1A and carries a cyclic RGD
epitope in the HI-loop of the fibre (Suzuki et al, 2001). This CRAd Cells were seeded at a density of 1.67  105 cells in a six-wells plate.
was propagated on A549 cells (American Type Culture Collection) After 24 h, cells were subjected to an IC10 or IC90 dose of
and purified using cesium chloride gradient banding and titrated doxorubicin. At 31 h or 6 days after adding doxorubicin, cells were
by end point limiting dilution on 293 cells (American Type Culture harvested and incubated in 300 ml propidium iodide (PI) solution
Collection). Doxorubicin was purchased from Pharmacia & (PBS supplemented with 0.05% PI, 0.1% Triton X-100, 0.1%
Upjohn (Woerden, The Netherlands) and cisplatin from Pharma- sodium citrate and 0.1% RNAse A; Sigma, St Louis, MO, USA) for
chemie BV (Haarlem, The Netherlands). 1 h at 41C. A minimum of 7000 cells was analysed by flow
cytometry on a FACScan (Becton Dickinson, Erembodegem-Aalst,
Belgium) and subsequently by ModFitLT cell cycle analysis
Cell viability assay software (Verity Software House, Topsham, ME, USA).
Cells were seeded in a 96-wells plate at a density of
5  103 cells well
1 and incubated for 24 h. Infections with a two-
fold dilution series of Ad5-D24RGD were performed in DMEM-F12 RESULTS
containing 2.5% FCS for 1 h. Serial dilutions of chemotherapeutic Combination of Ad5-D24RGD with cisplatin or
agents were added immediately, or after 24, 48 or 72 h in medium doxorubicin enhances the cytotoxic effect on OS cell lines
containing 10% FCS. Five to seven days poststart of treatment the
relative cell viability was measured by means of WST-1 conversion The combined effect of Ad5-D24RGD with cisplatin or doxorubicin
assay (Roche Diagnostics, Mannheim, Germany). WST-1 conver- was studied on the OS cell lines SaOs-2, MG-63 and U2OS. First,
sion was analysed by measuring the A450 using a Bio-Rad model IC50 values for single treatment with cisplatin, doxorubicin or

British Journal of Cancer (2006) 94(12), 1837 – 1844 & 2006 Cancer Research UK
 Chemo-gene therapy for OS
HCA Graat et al
1839
Ad5-D24RGD were determined by subjecting the cells to a range of Combination treatment of Ad5-D24RGD with cisplatin or
drug concentrations or CRAd MOI and measuring cell viability doxorubicin does not enhance the cytotoxic effect on OS
6 days later (Table 1). Next, combination effects were studied primary cell cultures
by simultaneous treatment in the range of 0.063 to 4-times the
individual IC50 (Figure 1A). Possible interactions between the To verify if the combination treatments also resulted in
drugs and Ad5-D24RGD were determined by CalcuSyn analysis enhanced cytotoxicity on primary OS samples, we used a panel
(Figure 1B). The average CI values from three independent of eight primary OS cell cultures (Table 2). All specimens were
experiments indicated that combined treatment with Ad5- diagnosed as high-grade OS. Five specimens were obtained from
D24RGD and doxorubicin was synergistic in SaOs-2 and U2OS patients that had not been subjected to chemotherapy; three
cells (mean CI values 0.55 and 0.81, respectively) and additive in samples (marked A) were taken after chemotherapy. Functional
MG-63 cells (mean CI ¼ 1.07). Combination treatment with p53 status differed considerably, ranging from deficient to wild-
cisplatin was on average synergistic in MG-63 and SaOs-2 cells, type activity. Marginal p53 activities in several samples
with mean CI values of 0.77 and 0.46, respectively, and antagonistic suggested heterogeneity. Interestingly, chemotherapy appeared
in U2OS cells (mean CI ¼ 1.22). Owing to variation between to select for more p53-deficient cell populations at least in one
individual experiments, CI values sometimes extended into a of the two cases. Limited amounts of available primary cells
different classification, suggesting, for example, an antagonistic precluded CalcuSyn analysis over a range of CRAd and drug
effect of CRAd plus doxorubicin on MG-63 cells or an additive concentrations. Therefore, cells were only treated with Ad5-
effect of CRAd plus cisplatin on U2OS cells. However, combination D24RGD, chemotherapeutic drug, or a combination of both at a
treatment was never less effective than either treatment alone. moderate toxic dose resulting in an estimated 20 – 60% cell kill.
Hence, in general, simultaneous treatment of OS cell lines with In this range, combination effects on OS cell lines were most

Translational Therapeutics
Ad5-D24RGD and chemotherapy yielded additive to synergistic evident (see Figure 1A). On different primary cell specimens, the
effects. doses used ranged from 0.1 to 3 PFU cell
1 Ad5-D24RGD, 0.6 to
1 mM doxorubicin and 3 to 18 mM cisplatin. Surprisingly, and in
contrast to the observations made on OS cell lines, combination
treatment of Ad5-D24RGD with doxorubicin or cisplatin did in
Table 1 IC50 values of Ad5-D24RGD, doxorubicin and cisplatin on OS most cases not result in a more pronounced cytotoxic effect
cell lines compared to the most effective single agent treatment (Figure 2).
Specimen OS-16 was the only exception where combination
Ad5-D24RGD Doxorubicin Cisplatin treatment was significantly better than either single agent
OS cell line (MOIa) (nM) (lM) treatment (Po0.05).
To investigate if combined treatment could be improved by
SaOs-2 8.573.3b 37716 2.370.8 delayed addition of chemotherapeutic agents, primary OS cells
MG-63 15.777.3 34712 0.970.2
were infected with Ad5-D24RGD; and cisplatin or doxorubicin
U2OS 1.171.2 2807190 5.673.0
were added 24, 48 or 72 h later. As shown in Figure 3, this did also
a
MOI ¼ multiplicity of infection; OS ¼ osteosarcoma. bData are means with standard not result in enhanced cell kill compared to monotherapy with
deviation from at least three independent experiments. Ad5-D24RGD or cisplatin or doxorubicin.

A Doxorubicin Cisplatin B 1.4 Ad5-∆24RGD with doxorubicin

100 100
80 80 1.2
% cell survival compared to nontreated control

60 60 1.0
SaOs-2 40 40 0.8
20 20
0.6
0 0
0.01 0.1 1 10 0.01 0.1 1 10 0.4
0.2
100 100
Cl values

80 80 0.0
SaOs-2 MG-63 U2OS
60 60
MG-63 40 40 1.4 Ad5-∆24RGD with cisplatin
20 20
0 0 1.2
0.01 0.1 1 10 0.01 0.1 1 10 1.0
100 100 0.8
80 80 0.6
60 60
0.4
U2OS 40 40
20 20 0.2
0 0 0.0
0.01 0.1 1 10 0.01 0.1 1 10 SaOs-2 MG-63 U2OS
Times average IC50 values OS cell lines

Figure 1 The oncolytic effect of Ad5-D24RGD in combination with doxorubicin or cisplatin on OS cell lines. Three OS cell lines (SaOs-2, MG-63 and
U2OS) were incubated at a concentration range of 0.063 to 4-times the average IC50 of each treatment agent. (A) Left panels represent cytotoxicity on OS
cell lines of Ad5-D24RGD (open diamonds), doxorubicin (open squares) as single agents or the combination of both (closed triangles). Right panels
represent cytotoxicity obtained with Ad5-D24RGD (open diamonds), cisplatin (open squares) or the combination (closed triangles). Cell survival was
expressed relative to nontreated controls. Data represent a typical experiment performed in triplicate. (B) Results from the combination experiments were
analysed by the CalcuSyn program. Data shown are mean CI values with s.d. from three independent experiments each performed in triplicate. CI values
below 0.9 were defined as synergistic, between 0.9 and 1.1 as additive and above 1.1 as antagonistic.

& 2006 Cancer Research UK British Journal of Cancer (2006) 94(12), 1837 – 1844
 Chemo-gene therapy for OS
HCA Graat et al
1840
Table 2 Characteristics of primary OS cells

Code Age M/F Diagnose p53 statusa (PG13-Luc/MG15-luc) Population doubling time (days)

OS-5A 7 F High grade OS 3.1 47

OS-6 25 M High grade OS 1.2 47
OS-8 6 M High grade OS 10.5 3.470.7
OS-11 16 F High grade OS 4.8 3.671.0
OS-11A 1.5 4.971.2
OS-12 13 F High grade OS 9.8 4.470.7
OS-12A 7.6 4.270.8
OS-16 15 F High grade OS 5.3 1.470.5
a
Functional p53 status was determined by measuring relative luciferase expression after PG13-Luc transfection compared to MG15-Luc transfection. Scores: ratio 0.5 – 2.0,
deficient; ratio 2 – 10, heterogeneous; and ratio410, functional. OS ¼ osteosarcoma.

120 Doxorubicin primary cell culture OS-16 and cell lines SaOs-2, MG-63 and
U2OS, where combination treatment had shown additive or
% cell survival compared to nontreated control

100
synergistic effects. Cells were infected with 50 PFU cell
1 Ad5-
80
D24RGD and one hour after infection cells were cultured in the
60 presence or absence of a subtoxic dose (IC10) of doxorubicin. The
Translational Therapeutics

40 next day, CRAd genome copy numbers were determined by

20 quantitative PCR analysis. As can be seen in Table 3, doxorubicin
0 decreased viral replication in OS-11 and OS-12A cells by 2.5- and
OS-5A OS-6 OS-8 OS-11 OS-11A OS-12 OS-12A OS-16 five-fold, respectively. In contrast, in OS-16, SaOs-2, MG-63 and
120 Cisplatin U2OS cells viral replication was not significantly affected. In fact,
viral replication in SaOs-2 cells was not even inhibited in the
100
presence of a toxic concentration of doxorubicin.
80
60
Doxorubicin increases the OS cell population in G2/M
40 phase of the cell cycle, but not in OS-11 and OS-12A cells
20
Towards explaining the different response of most primary OS
0
cultures vs OS-16 and OS cell lines, we investigated their cell
OS-5A OS-6 OS-8 OS-11 OS-11A OS-12 OS-12A OS-16
Primary OS cells growth rate. SaOs-2, MG-63 and U2OS OS cell lines all exhibited a
fast proliferation rate with a population doubling time of 0.7 – 0.8
Figure 2 The cytotoxic effect of Ad5-D24RGD with doxorubicin or days (data not shown). In contrast, most primary OS specimens
cisplatin on primary OS cell cultures. Eight primary OS cell cultures were proliferated much slower (doubling times ranging from 3.4 to 47
subjected to concentrations of Ad5-D24RGD (white bars), doxorubicin days; see Table 2). Osteosarcoma-16 cells were the exception, with
(hatched bars; upper panel) or cisplatin (hatched bars; lower panel)
resulting in 20 – 60% cell kill or to a combination of CRAd plus a doubling time of only 1.4 days. On the basis of this observation
chemotherapeutic drug (black bars). Relative cell survival compared to and the known effect of doxorubicin in arresting cells in the G2
nontreated cultures was measured by WST-1 conversion assay. Data phase of the cell cycle, which has been reported to enhance viral
shown are mean values with s.d. from experiments performed in triplicate. replication (Bernt et al, 2002), we investigated whether a G2 cell
cycle arrest occurred in OS cell lines and primary samples treated
with doxorubicin.
Within 31 h, doxorubicin induced a G2 cell cycle arrest in SaOs-
To further corroborate these findings, OS cell lines and a 2, MG-63 and U2OS cells (Figure 5). This was already evident when
random selection of the primary OS panel were subjected to cells were treated with an IC10 dose of doxorubicin and was more
approximate IC10 (subtoxic) dose of cisplatin or doxorubicin pronounced at an approximately IC90 dose. In the same time,
combined with an approximate IC70 (toxic) dose of Ad5-D24RGD doxorubicin also caused a marked increase in the proportion of
(Figure 4). On OS cell lines, a subtoxic dose of cisplatin or OS-16 cells in the G2/M phase of the cell cycle (Figure 6). In
doxorubicin did not reduce the Ad5-D24RGD cytotoxic effect. In contrast, the cell cycle status of OS-11 and OS-12A cells was hardly
contrast, on most primary specimens the cytotoxic effect of Ad5- affected. This was even the case after extended culture for 6 days,
D24RGD was inhibited by the low dose of cisplatin or doxorubicin. that is, for more than one population doubling time (Figure 6). To
Again, this significant antagonistic effect (Po0.05) was seen in all investigate if OS-11 and 12A cells would require higher doses of
primary OS cell cultures tested, except OS-16. doxorubicin to induce G2 arrest, cells were also treated with
doxorubicin at concentrations up to 1.5 mM (approximate IC80).
Under these conditions, we observed even a decreased G2-phase
Combination with doxorubicin inhibits viral replication in cell population in favour of the G1 cell cycle phase (data not
OS-11 and OS-12A cells, but not in OS-16, SaOs-2, MG-63 shown). Thus, an increased cytotoxic effect of combination
and U2OS cells treatment of Ad5-D24RGD with doxorubicin on OS cells appeared
We postulated that the different outcome of the tested combina- to correspond with susceptibility to doxorubicin-induced G2 cell
tion treatments might be due to a different effect of chemo- cycle arrest.
therapeutic agents on viral replication. To test this hypothesis,
we measured viral replication in the presence or absence of DISCUSSION
doxorubicin. This was performed on primary cell cultures OS-11
and OS-12A, where we had observed an adverse effect of Combination treatment of chemotherapy and virotherapy holds
doxorubicin on Ad5-D24RGD-mediated cytotoxicity, and on promise as a new strategy for cancer treatment (Khuri et al, 2000;

British Journal of Cancer (2006) 94(12), 1837 – 1844 & 2006 Cancer Research UK
 Chemo-gene therapy for OS
HCA Graat et al
1841
24 h 48 h 72 h
% cell survival compared to nontreated control
120 120 Doxorubicin 120
100 100 100
80 80 80
60 60 60
40 40 40
20 20 20
0 0 0
OS-6 OS-8 OS-11 OS-12A OS-6 OS-8 OS-11 OS-12A OS-6 OS-8 OS-11 OS-12A

120 120 Cisplatin 120

100 100 100
80 80 80
60 60 60
40 40 40
20 20 20
0 0 0
OS-6 OS-8 OS-11 OS-12A OS-6 OS-8 OS-11 OS-12A OS-6 OS-8 OS-11 OS-12A
Primary OS cells
Figure 3 Effect of delayed doxorubicin or cisplatin treatment after infection with Ad5-D24RGD on primary OS cell kill. Primary OS cells were infected

Translational Therapeutics
with Ad5-D24RGD and 24, 48 or 72 h later cells were treated with doxorubicin or cisplatin (black bars). Controls treated only with virus or
chemotherapeutic drug are shown by white and hatched bars, respectively. Relative cell survival was measured by WST-1 conversion assay. Data shown are
mean values with s.d. from experiments performed in triplicate.

Doxorubicin Table 3 Ad5-D24RGD viral replication in doxorubicin-treated OS cells

120 120 as determined by quantitative PCR analysis
100 100
% cell survival compared to nontreated control

80 80 Average
Genomes  105 genomes  105 +dox/ ratio: +dox/
60 60 OS cells
doxa +dox
dox
dox (7s.d.)
40 40
20 20 5.8 3.5 0.6
OS-11 3.3 0.9 0.3 0.4 (70.2)
0 0 4.1 1.1 0.3
SaOs-2 MG-63 U2OS OS-8 OS-11 OS-12 OS-12A OS-16
Cisplatin 6.5 1.4 0.2
120 120 OS-12A 1 0.2 0.2 0.2 (70.04)
100 100 3.4 0.5 0.1
80 80
17 16 0.9
60 60
OS-16 8.9 14 1.6 1.3 (70.3)
40 40 75 94 1.3
20 20
0 0 2.1 2.9 1.4
SaOs-2 MG-63 U2OS OS-8 OS-11 OS-16 SaOs-2 5.5 6.4 1.2 1.1 (70.3)
11 8 0.7
Figure 4 Effect of subtoxic dose of doxorubicin or cisplatin on the
oncolytic effect of Ad5-D24RGD on OS cell lines and primary OS cells. 1.6 3.4 2.1
Osteosarcoma cell lines and primary OS cell cultures were treated with MG-63 7.7 7 0.9 1.2 (70.8)
approximate IC70 of Ad5-D24RGD (white bars) and the approximate IC10 11 6.2 0.6
of doxorubicin or cisplatin (hatched bars) or a combination (black bars).
Relative cell survival was measured by WST-1 conversion assay. Data 20 15 0.8
shown are mean values with s.d. from experiments performed in triplicate. U2OS 96 127 1.3 1.0 (70.3)
87 72 0.8

2.1 4.8 2.3

Lamont et al, 2000; Reid et al, 2001). Here, we investigated the SaOs-2b 5.5 5.1 0.9 1.8 (70.8)
effect of the combination of Ad5-D24RGD with chemotherapy for 11 25 2.3
OS. In order to detect possible interactions between Ad5-D24RGD a
and chemotherapeutic drugs, Ad5-D24RGD-infected OS cells were The number of adenovirus genomes in cells cultured in the absence (
dox) or
cultured in the continuous presence of doxorubicin or cisplatin. presence (+dox) of a subtoxic dose doxorubicin was determined in three
independent experiments and the average ratio and s.d. was calculated. bSaOs-2
We found that OS cell lines are killed more effectively by a cells treated with a toxic dose of doxorubicin. PCR ¼ polymerase chain reaction;
combination of Ad5-D24RGD with doxorubicin or cisplatin than OS ¼ osteosarcoma.
by either single agent treatment. Combined effects were pre-
dominantly additive to synergistic. This is in line with observa-
tions reported by others on different cancer types (Heise et al,
1997, 2000; You et al, 2000; Li et al, 2001; Yu et al, 2001; Portella Only one of the eight tested primary OS cultures showed a
et al, 2002). Surprisingly, however, combination treatment did not significantly increased cytotoxicity following combination treat-
lead to a similar more effective cell kill on primary OS cell cultures. ment. Delaying the addition of chemotherapeutic drug after virus

& 2006 Cancer Research UK British Journal of Cancer (2006) 94(12), 1837 – 1844
 Chemo-gene therapy for OS
HCA Graat et al
1842
Control + IC10 doxorubicin + IC90 doxorubicin

360 19% 360 23% 360 79%

270 270 270
SaOs-2
180 180 180
90 90 90
0 0 0
0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250

400 10% 400 17% 400 83%

300 300 300
MG-63
200 200 200
100 100 100
0 0 0
0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250
500 500 500
400 9% 400 37% 400 80%
Translational Therapeutics

U2OS 300 300 300

200 200 200
100 100 100
0 0 0
0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250
Propidium iodide fluorescence intensity
Figure 5 Effect of doxorubicin on cell cycle profile of OS cell lines. SaOs-2, MG-63 and U2OS cells were cultured for 31 h in medium containing IC10 or
IC90 doxorubicin as indicated. DNA histograms were made by PI staining and FACS flow cytometry. DNA histograms were analysed by ModFitLT cell cycle
analysis software. Percent cells in G2/M phase of the cell cycle are indicated in the panels.

31 h 6 days
Control + IC10 doxorubicin Control + IC10 doxorubicin
1600 1600 1600 1600
G2: 10% G2: 5% G2: 8% G2: 6%
1200 1200 1200 1200

OS-11 800 800 800 800

400 400 400 400

0 0 0 0
0 50 100 150 0 50 100 150 0 50 100 150 0 50 100 150

G2: 18% 800 G2: 18% 800 G2: 5% G2: 5%

800 800
600 600 600 600
OS-12A
400 400 400 400
200 200 200 200
0 0 0 0
0 50 100 150 0 50 100 150 0 50 100 150 0 50 100 150

G2: 22% 800 G2: 56% 800 G2: 6% 800 G2: 21%
800
600 600 600 600
OS-16
400 400 400 400

200 200 200 200

0 0 0 0
0 50 100 150 0 50 100 150 0 50 100 150 0 50 100 150
Propidium iodide fluorescence intensity
Figure 6 Effect of doxorubicin on cell cycle profile of primary OS cells. Osteosarcoma-11, OS-12A and OS-16 cells were cultured in medium containing
IC10 doxorubicin for 31 h or 6 days as indicated. DNA histograms were made and analysed as described in the legend to Figure 5.

British Journal of Cancer (2006) 94(12), 1837 – 1844 & 2006 Cancer Research UK
 Chemo-gene therapy for OS
HCA Graat et al
1843
infection for up to three days did not improve cell killing. This unresolved adverse effect of chemotherapy on CRAd replication in
suggested that the inhibitory effect of chemotherapy on virother- the absence of G2 cell cycle arrest.
apy in primary cells was unrelated to interference with early steps The markedly different response of OS cell lines and primary OS
of virus infection. specimens to combination treatment also raises the question which
Our findings raise the question why OS cell lines and primary of these cells are more relevant to predict efficacy of combination
cells responded differently to combination treatment. To answer treatment in the clinic. Although direct comparative data are not
this question we need insight into the mechanism of synergy available, we regard our findings obtained on primary specimens
between chemotherapy and virotherapy. Two different, not as more relevant predictors for clinical outcome than the
mutually exclusive mechanisms have been proposed to explain observations made on cell lines. This is based on the fact that
combination effects of CRAds and chemotherapy. On one hand, it the cytotoxic effects of doxorubicin and cisplatin on primary
has been postulated that CRAds enhance the efficacy of tumour cultures were already shown to correlate well with results
chemotherapy through viral E1A expression rendering tumour gathered in phase II clinical trials (Fridborg et al, 1999).
cells more susceptible to DNA damaging agents (Sanchez-Prieto Furthermore, it is known that the vast majority of human tumour
et al, 1996). On the other hand, it was reported that adenoviral cells in vivo are in a quiescent rather than in a fast proliferating
replication is increased in the G2 phase of the cell cycle state (Weisenthal, 1991, pp 103 – 147). In this respect, the slow in
(Steinwaerder et al, 2000; Bernt et al, 2002). Chemotherapeutic vitro growth rate for primary OS cells found herein is more
agents that induce a G2/M cell cycle arrest were shown to enhance comparable with the reported growth rate for OS cells in vivo than
viral replication, whereas an induction of a G1 cell cycle arrest that of the tested OS cell lines (Band and Kocandrle, 1975). This
poorly supported viral replication (Bernt et al, 2002). In our study, makes our observation that combination treatment was not
enhanced efficacy of the Ad5-D24RGD CRAd in combination with successful against slowly dividing OS cells particularly relevant.

Translational Therapeutics
chemotherapy appeared to correlate with susceptibility to chemo- Our observations described herein should not be interpreted as a
therapy-induced G2 cell cycle arrest, supporting at least the second discouragement for combination treatments with CRAds and
explanation for synergy between CRAd-induced oncolysis and chemotherapeutic agents altogether. It should be emphasised that
chemotherapy. This suggests that differential susceptibility of OS Ad5-D24RGD killed primary OS cells very efficiently and that this
cell lines and primary samples to doxorubicin or cisplatin could CRAd did not hamper the effect of the applied chemotherapy
have dictated a different response to combination treatment. against primary OS cells. Hence, treating OS patients with CRAds in
Differential susceptibility of cell lines and primary samples to the course of continued chemotherapy is not expected to affect the
chemotherapy is not uncommon. For example, the activity of standard treatment. However, chemotherapy did reduce the efficacy
topotecan, irinotecan and SN-38 against cancer cell lines was of CRAd treatment. Therefore, we propose that if combination
shown to differ from their activity against corresponding primary treatment of OS with CRAds and chemotherapy is considered, the
samples (Jonsson et al, 2000). As chemotherapy-induced G2 cell virotherapy and chemotherapy agents should best be administered
cycle arrest is more likely to occur in fast than in slowly dividing in intermittent administration schemes. Finally, in general, our
cells and OS cell lines had a much higher cell division rate than findings underscore the importance of preclinical testing of new
primary OS cells, the former cells may be more susceptible to G2 genetic anticancer agents and treatment regimens on primary
cell cycle arrest and thereby to enhancement of CRAd efficacy by human cancer specimens, because as shown herein the outcome
chemotherapy. In line with this reasoning, the only primary can be entirely different from results obtained on cell lines.
sample that was susceptible to combination treatment (OS-16)
proliferated markedly faster than any of the other primary cell
cultures. However, slowly dividing primary cell cultures did also ACKNOWLEDGEMENTS
not arrest in G2 phase when exposure to doxorubicin was
prolonged to encompass more than a population doubling time. We thank Dr JE Carette for helpful discussions. Ad5-D24RGD was
Moreover, while resistance to chemotherapy due to low cell kindly provided by Dr R Alemany (Gene Therapy Center, UAB,
division rate may explain a lack of synergy in primary OS cells, it Birmingham, AL, USA). M Adhiambo Witlox is supported by the
does not explain why chemotherapy even counteracted virotherapy Netherlands Organization for Scientific Research (NWO-AGIKO),
in many samples. Our findings suggest a delicate balance between, Frederik HE Schagen by the VU University Stimulation Fund
on one hand, a favourable role of chemotherapy-induced G2 cell (USF) and Victor W van Beusechem by a fellowship of the Royal
cycle arrest on viral replication and, on the other hand, a so far Netherlands Academy of Arts and Sciences (KNAW).

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