(CANCER RESEARCH57. 3200-3207. August I. 19971
Antisense Oligonucleotides Specific for Transforming Growth Factor
Growth of Malignant Mesothelioma Both in Vitro and in Vivo'
@2
Inhibit the
Amanda L. Marzo,2 David R. Fitzpatrick, Bruce W. S. Robinson, and Bernadette Scott
The University
of Western Australia
Department
of Medicine,
Queen Elizabeth
II Medical
B. W. S. R., B. SI. and the Transplantation Biology Unit, Queensland Institute ofMedical
Centre, Verdum Street, Nedlands, 6008 Perth, Western Australia,
ABSTRACT
Transforming
growth factor
@3
(TGF-@) is a potent growth-regulatory
many types of cells. High levels of TGF-f3 are produced by several human
and mouse malignant mesothelloma (MM) cell lines, and it is known to act
as a growth factor for these cells. Antisense oligonucleotides (ODNs),
against specific TGF-fJ mRNA, were used to block TGF-fi pro
duction from MM cells in vitro and in vivo.
TGF-@3antisense ODNs were encapsulated in liposomes and transfected
into MM cells or delivered Intratumorally. TGF-fi2 mRNA levels, assessed
by semiquantitative PCR, and TGF-Ø2 protein secretion were reduced
after
TGF-fi2
antisense
ODN transfection.
MM cell proliferation,
assessed
by tritiated thymidine uptake, was specifically Inhibited by both TGF-@1and TGF-@32-speclfic antisense ODNs. In vivo administration
of TGF-@2
antisense ODNs, delivered locally, reduced tumor growth. These data
show that the blockade of TGF4J2 within this tumor reduces tumor
growth
and
raises
useful as a therapy
the possibility
that
TGF432
antisense
ODNs
may
(A. L M.,
biology, clinical features, histopathology, and immunobiology (16),
provides an opportunity for evaluating the role of TGF-@ in tumori
genesis as well as its role in modulating the immune response to
tumor.
Antisense ODNs can block the translation of particular gene prod
ucts within cells and represent a unique method of specifically inhib
iting the effects of target proteins in cells, including these cells
producing intracrine-acting proteins (17). This inhibition by antisense
ODNs relies on the ability of an ODN to bind to a complementary
mRNA sequence and prevent translation of the mRNA. Antisense
ODNs may also interfere with the expression of the targeted sequence
by other mechanisms (18—20).
To test whether TGF-j3 promotes tumor growth and prevents de
velopment of an antitumor response, we added TGF-f3 antisense
ODNs to MM tumor cells both in vitro and in vivo. We provide
evidence that TGF-@ antisense ODNs do inhibit MM tumor growth
and suggest that TGF-@ has a role in MM tumorigenesis.
and immunomodulatory cytokine that exerts a diverse range of effects on
targeted
Australia
Research, 300 Herston Road, 4006 Herston, Queensland, Australia ID. R. F.]
be
for this disease.
INTRODUCTION
MATERIALS
AND METHODS
TGF-/33 represents a family of multifunctional polypeptides that
elicit different responses in different cell types including growth,
differentiation, and morphogenesis (1 , 2). In vertebrates, at least five
different forms have been identified (TGF-@3l to TGF-(35), all of
which form homodimers of -‘-25kDa. TGF-(3 signals through a
heteromeric complex of type I and type II receptors (3, 4). The type
II receptor directly binds ligand but is incapable of mediating TGF-j3
responses in the absence of a type I receptor (5). TGF-j3 is a complex
modulator of cell growth, generally inhibiting the growth of hemato
poietic cells while stimulating the growth of mesenchymal cells (6). In
the progression of some transformed cells to malignancy, TGF-@ may
switch from mediating growth inhibition to growth stimulation (7).
The role of TGF-@3as a suppressor of immune responses is also well
documented (8). Interestingly, TGF-j3 may play a role in determining
the nature of the immune response to antigen. It has been reported that
TGF-@ can promote the development of a T helper-2 phenotype in T
cells in BALB/c mice infected with Leishmania braziliensis and that
TGF-f3 strongly inhibits the differentiation of naive CD4@ T cells
toward T helper-I phenotype (9, 10).
MM is an aggressive tumor of the serosa and pleura induced by
exposure to asbestos. Significant levels of TGF-(3 are produced by
some human MM cell lines (11—13),and a role for TGF-@ as a direct
or indirect factor in MM growth and tumorigenesis has been sug
gested (14, 15). The development of a syngeneic mouse model of
MM, which faithfully reproduces its human counterpart in terms of
Animals. CBA/CAH (H@2k) mice (SPF; female; 6-8 weeks old) were
obtained from the Animal Resources Centre (Perth, Australia) and maintained
under standard conditions in the University Department of Medicine animal
holding area. All animal experimental procedures were approved by the
University of Western Australia Animal Welfare Committee and followed the
National
Health and Medical
Research
Council
Thecostsof publicationof thisarticleweredefrayedin partby the paymentof page
charges. This article must therefore be hereby marked advertisement
18 U.S.C. Section 1734 solely to indicate this fact.
in accordance with
I Supported by the National Health and Medical Research Council ofAustralia
gentamicin
(David Bull Labs, Melbourne,
Australia),
2 To
whom
requests
for
reprints
should
be
addressed.
Phone:
061-8-9-346-3259;
Fax:
061-8-9-346-2816; E-mail: [email protected].
3 The abbreviations used are: TGF-@, transforming
mesothelioma;
ODN, oligonucleotide;
SQ-PCR,
growth factor
semiquantitative-PCR;
modeoxyuridine; ilL, tumor-infiltrating lymphocyte.
@;
MM,
malignant
and 5% fetal bovine
serum (Life Technologies, Inc.).
ODNs. Antisense TGF-f3l, TGF-@32,and sense ODNs were synthesized
commercially by standard techniques (Bresatec Ltd., Adelaide, Australia).
Modified phosphodiester ODNs, with substitution of nonbridging oxygen by a
sulfur producing a phosphorothioate linkage, were used in in vivo experiments,
because unmodified
phosphodiester
in the presence of serum (21).
ODNs
have a half-life
of less than 15 mm
[3HlThymidine Incorporation. AC29 cells were seeded into triplicate
wells of a 96-well flat-bottomed plate (Nunc, Roskilde, Denmark) at a density
of l0@cells/well and incubated at 37°Cfor 24 h. Antisense ODNs were
transfected
into AC29
cells
using
N-[l-(2,3-dioleoyloxyl)propyl]-N,N,N-tri
methylammoniummethyl sulfate (Boehringer Mannheim, Sydney, Australia) at
a ratio of 5:1 as described by the manufacturer. Briefly, ODNs were incubated
with liposomes at room temperature for 10 mm and then made up to the
appropriate concentration (0.1 @g/wel1
antisense ODN) in RPMI 1640 con
at 65°Cfor 30 mm to macti
vate exonucleases). DNA/liposome formulations were added to the seeded
AC29 cells to a total volume of200 id/well. Proliferation was assessed on days
1, 2, 3, and 4 by [3H]thymidine(Amersham, Sydney, Australia) incorporation.
[3H]Thymidine
and the
Medical Research Fund of Western Australia.
for Exper
Tumor Cell Lines. The derivation and basic characterization of the AC29
murine MM cell line used in this study has been described previously (14).
AC29 cells (CBA-derived) were maintained in RPM! 1640 (Life Technologies,
Inc.) supplemented with 20 mM HEPES, 5 X l0@ M 2-mercap
toethanol, I x l0@units/liter penicillin (CSL, Perth, Australia), 50 mg/liter
miming 2% fetal bovine serum (heat-inactivated
Received I 2/1 3/96; accepted 5/29/97.
Recommendations
iments in Animals.
was added
at 1 MCi/well
12 h before
harvesting
cells.
Cells
were harvested onto glass fiber filter papers using a PHD cell harvester
(Cambridge Technology, Inc., Boston, MA), and thymidine incorporation was
measured by liquid scintillation.
TGF4J1and -@32
ELISA. Supernatants
fromcells incubatedwithsense
BrdUrd, bro
and antisense TGF-@l and-f32 ODNs were collected and assayed for total
3200
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INfflB@ON OF TUMOR GROWTH BY ANTISENSE TGF-@
Table1 Sequenceof PCR
primersGene
were exposed for 0.5—Imn and analyzed using an ImageQuant densitometer
(Molecular Dynamics, Sunnyvale, CA). The results were expressed relative to
the 13-actinsignal in each sample.
TGF-@5
5'-AGAGTACTACGCCAAGGAGGT--3'
i.p. Effects of ODNs on Survival of Animals. Groups of 10 mice were
3'TGF-@l TGF-@6Da
U―
5 -AGGAGGGCAATAACATTAGCATGF-138D
3'TGF-@9U
5‘
-GAGGTCACCCGCGTGCTAATGinoculated i.p. with 2 X l0@untransfected AC29 cells or AC29 cells trans
5'-GGCCAGGACCTTGCTGTACTG-3'f3-actin
fected 24 h before injection with either antisense TGF-@3l,TGF-(3 2, sense
HBA1D
3'HBA2U
5' -CGTGACATTAAGGAGAAGCTGTGCTGF-f3(complementary
sequence to the antisense TGF-@3lODN), or nonsense
3'a
5‘
—CTCAGGAGGAGCAATGATCTTGATODN [a random mixture of all four nucleotides from the TGF-@32sequence
D, downstream primer.
(Table 2)]. ODNs incorporating two different backbones (phosphodiester or
b U, upstream primer.Table
phosphorothioate) were tested. Clinical signs were monitored daily until ter
mination as described previously (25).
PrimerLocation
SequenceTGF-@2
@
ODNsODNBackbone
Intratumoral
2 Sequence of
SequenceAntisense
3'AntisenseTGF-@2D/Ta
3'Nonsense TGF-@1DIT
3'Sense TGF-@32T
5, -GATCTGGCCGCGGAT-
TGF-@lD/T
3'Sense
5‘
-TCCCCCATGCCGCCC-
5 ‘
-GGGCGGCATGGGGGA-
5‘
-GGCTCGATTCGGGAC-
TGF-@2
5 ‘
-TAGGCGCCGGTCTAG-
Antisense
TGF-@2Dir
a D,
b T-C,
phosphodiester
C-S
propyne
backbone;
T-C@'
pyrimidine-modified
BrdUrd
3'
5'-GA656GG55G5GGA6-3'
T, phosphorothioate
backbone.
phosphorothioate
backbone,
where
6 =
CS
propyne and 5 = CS propyne.
TGF-@protein (latent and active) as described by Danielpour et a!. (22) with
slight modifications. Briefly, Nunc maxisorb microtiter plates were coated
with a monoclonal mouse anti-TGF-@l antibody (lDll.16; Genzyme, Cam
bridge, MA) in carbonate buffer overnight at 4°C.The capture antibody was a
chicken anti-TGF-@antibody (R&D Systems, Inc., Minneapolis, MN) and was
detected using an antichicken alkaline phosphatase conjugate (Sigma, Sydney,
Australia). Samples were assayed for TGF-(32 protein using a commercial
immunoassay kit (R&D Systems, Inc.). The lDll antibody has a maximal
cross-reactivity with TGF-f33 of 5%, and 60 @g/m of this antibody are
required to neutralize TGF-f32 compared with 0.6 @gfor TGF-f3l. The
TGF-132assay has less than 0.1% reactivity with TGF-(31and TGF-@33(R&D
Systems, Inc.).
SQ-PCR. In vitro, AC29 cells were seeded into triplicate wells of a 96-well
flat-bottomed plate at a density of iO@cells/well and incubated overnight at
Treatment.
To determine the in vivo proliferation of tumor cells,
mice were injected i.p. with 200 @.tg
of BrdUrd 16 h before sacrifice (26).
Confocal Laser Scanning Microscopy. Cryostat sections ([email protected])
were fixed at 4°Cin 2% cold paraformaldehyde for 15 mm, washed in PBS
three times for 5 mm, and incubated in lN HC1for 10 mm at 60°C.Sections
were then washed in 0.1 M Tris (pH 8.5) for 5 mm and incubated with the
anti-BrdUrd-FITC (Becton Dickinson, Sydney, Australia) at 1:10 in 0.1 MTris
(pH 8.5) and 0.1% Tween 20 overnight at 4°C.Sections were washed five
times for 10 mn and then mounted with Immu-mount (Shandon, Pittsburgh,
PA). A Bio-Rad 1000 MRC system fined with a krypton/argon-ion laser and
built around a Nikon Diaphot 300 mcroscope was used. The 488 nm wave
length line was used for excitation of FITC, and the images were captured with
Comos software. Three sections from each time point for both antisense and
nonsense sections were stained and investigated on the same day. A X20
plano-apochromate Nikon objective was used (numerical aperture, 0.75). From
each section, fields of view from both the center of the tumor and the periphery
were chosen, and a stack of 14 optical sections was scanned with a z increment
of 2 @imand then projected to produce a picture size of 384 X 512 pixels.
Images were further analyzed in Corelphotopaint, and slides and photomicro
graphs were produced using Microsoft Powerpoint.
TIL Isolation
and Analysis.
ilLs
were generated by methods published
tumor cells grown in vitro and tumors excised from mce, reverse-transcribed
into cDNA using random primers and avian myeloblastosis virus reverse
using Lysis II software.
transcriptase,
@
Groups of six mice
previously (27, 28) with some modifications. Briefly, tumors were harvested
on days I 1, 15, 19, and 23 after s.c. injection of AC29 cells. Mice received
antisense and nonsense ODN intratumoral injections from day 11 until the
mice were euthanized. Tumors were minced and then digested with constant
stirring in 25 ml of RPMI 1640 containing 2000 units of DNase (Sigma) and
1 mg/mI collagenase (Sigma) for 1.5 h at room temperature. The cell suspen
sion was washed, filtered through nytex, and analyzed for surface marker
expression by flow cytofluorometric analysis on a Becton Dickinson FACScan
37°C.ODNs were added to the seeded plate, and 12 h later, triplicate wells
were pooled, and mRNA was extracted. For in vivo analysis, total RNA was
extracted from tumors excised from mce that had been previously treated with
either antisense TGF(3-2ODNs or a nonsense ODN intratumorally by guam
dinium isothiocyanate-phenol extraction using RNaz01THB(Bresatec) as de
scribed previously (23). Briefly, total RNA (25 @g)
was extracted from both
@
Effect of TGF-@2 on Tumor Growth.
were inoculated s.c. with 2 X l0@AC29 cells. Once tumors became palpable
(8 mm3),mice received 2 @.tg
intratumorally of either antisense TGF-@32
ODN,
a nonsense ODN, or liposomes alone. Before intratumoral injections, ODNs
were encapsulated into liposomes as detailed previously.
and amplified
by PCR using TGFj3-l-
and TGF-@32-specific
primers (Table 1). Gene-specific intron-spanmng primers were designed using
the Oligo program (Bresatec) and obtained commercially (Bresatec). PCR
reactions for each set of primers were performed separately in a total volume
of 25 @.d
containing 1X PCR buffer, 1 unit of Tth-plus DNA polymerase
(Biotec International, Perth, Australia), 100 ng of each primer, 1.5—2.0
mM
MgC1,200 @.LM
dNTP, and 0.5 @.tg
of the previously diluted reverse transcrip
tion reaction. Amplifications were performed in a HBTRI thermal cycler
(Hybaid, Middlesex, United Kingdom) by using the following conditions:
cycle 1, denaturing at 94°Cfor 5 mn, annealing at 55°Cfor 1 mm, and
extension at 72°Cfor 2 mm; cycles 2—26
(amplification cycles for tumor cells
grown in vitro) and cycles 2—32(amplification
cycles for tumors grown in
vivo), denaturing at 94°Cfor 30 s, annealing at 55°Cfor 1 mm, and extension
at 72°Cfor 2 mn; and cycle 27 or 33, denaturing at 94°Cfor 30 s, annealing
at 55°Cfor 1 mm, and extension at 72°Cfor 10 mn. To ensure that reverse
transcriptase efficiencies were comparable between test groups, @3-actincDNA
was coamplified with TGF-(3 in the same tube, and the amplification was
limited to 27 cycles (13).
Quantification
of PCR Products. The resulting PCR products were sep
arated on a 2% agarose gel and transferred to nylon membranes as described
Immunohistochemistry.
Tumor, spleen, and lymph node samples from
euthanized mce were taken at days I 1, 15, 19 and 23 from mice receiving
160
140
@120
El TGF-@i
.
TGF-@2
I
UPOSOMES
ONLY
@1oo
U
;80
0
60
0
1
2
3
4
Time (days)
Fig. I. Proliferation response of AC29 MM cells to antisense TGF@ ODNs. Untrans
previously (24). The membranes were hybridized with biotinylated TGF-@3l- fectedAC29MMcellsor
cellstransfectedwithantisenseTGFf3ODNsor senseTGF@
and TGF-132-specific probes, and the binding was visualized using streptavi
ODNs (0.1 g.@g/well)
were incubatedfor 1, 2, 3, and 4 days and assayedfor cell
din-peroxidase-coupled enhanced chemlumnescence (Amersham). Films proliferation. Results are reported as a mean of triplicate observations ±I SE.
3201
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INH@@ONOF TUMORGROWTHBY AN'FISENSE
TGF-@
A
TGF- f31
2
@
15
TGF432
0.7
2.5
@
B
U UP080MESOt'LY
TGF.@2
0.6
TGF-@1
U [email protected]'LY
D
TGF-@2
D
TGF.@1
U
OS
0.5
Fig. 2. Effect of antisense TGF-@ ODNs on
0
targetmRNAand proteinproduction.AC29cells
0UG0NucLE0'nDESADDED
ouGoNucLEo@nDEsADDED
were transfected with antisense or sense ODNs or
left untransfected and then assayed for the produc
tion of TGF-f31(A) or TGF-@2(B) mRNAby
SQ-PCR. The result is expressed as a ratio relative
to the level of /3-actin. Supernatants from these
cultures were assayed at three time points for
TGF-131(C) or TGF-@2(D) by ELISA.The data
are representative of three separate experiments.
D
C
70
I0
S
.@60
40
20
10
0
DAYS
intratumoral injections of ODNs. Tissues were immersed in OCT (Miles, Inc.,
Elkhart, IN) for 5 mn, frozen on dry ice, and stored at —80°C
until analysis.
Cryosections (10-sm thick) were cut at —18°Cand transferred to tissue-tek
slides. Slides were fixed in i% paraformaldehyde for 30 mm, blocked with 1%
H202 for 5 mn, and immunolabeled in a three-step protocol using monoclonal
antibodies and biotin/streptavidin
conjugates. Monoclonal antibodies specific
for murine CD4@ cells (rat anti-CD4; GK1.5), CD8@ cells (rat anti-CD8;
53.6.72), macrophages (rat antimacrophage; F4/80), and class II (MS.! i4)
were used. All incubations were performed at room temperature for 45 ruin
with primary antibody and 30 mn with each of the conjugates, with three
5-mn washesin PBSin between.IncubationwithdiaminobenzidinelH2O2
for
20 mn at room temperature was followed by counterstaining with hematox
ylin. Sections were mounted in glycerol gelatin (Sigma).
RESULTS
Effects of TGF-@3 Antisense ODNs in Vitro. TGF-f3 antisense
ODNs reduced the proliferative capacity of the AC29 MM cell line in
10
w
>
...1—.
--s-
-I
8
Cl)
6
UPOSOMES
ONLY
- NONSENSE
U
TGF@2-C
-I
4
4
z
4
2
0
z
0
24
26
DAYS
28
30
POST
32
34
36
38
40
vitro. When phosphodiester
in vitro meant
CHALLENGE
Fig. 3. Effect of antisense TGF(3 ODNs on the survival of tumor-bearing mice. Mice
received AC29 MM cells (2 X l0@ cells) transfected with TGF(3 ODNs (0.1 @ag/
2 X lO'cells) i.p. Control mice received AC29 MM cells (2 X 10@cells) that were either
untransfected or transfected with a nonsense ODN.
antisense
ODNs were added to MM cells
at the initiation of the culture period and proliferation was determined
over 4 days, proliferation was specifically inhibited up to 50% by
either TGF-31 or TGF-@32 antisense ODNs and was particularly
marked by days 3—4of the experiment (Fig. 1).
To determine whether this inhibition of MM cell proliferation
corresponded to a reduction in the amount of TGF-@ produced,
TGF-@ mRNA was assessed using SQ-PCR procedures standardized
against the level of (3-actin. In these experiments, phosphorothioate
derivatives were used, because having a longer half-life in serum, they
would be used in in vivo experiments (21). Twelve h after the addition
of TGF-f31 antisense ODNs, TGF-@1 mRNA was reduced to unde
tectable levels (Fig. 2A). This reduction was specific, because TGF-@2
mRNA levels were unaffected, and the sense ODNs reduced TGF-(31
mRNA expression only slightly compared to that of untransfected
controls. Antisense TGF-j32 antisense ODNs also reduced TGF-j32
mRNA up to 70% of that in untransfected controls and 50% of that in
specificity controls (i.e., the sense and antisense TGF-@1 ODNs; Fig.
2B). Also, TGF-@ protein production was measured by determining
the amount of TGF-@1 or (32 in the MM culture supernatant by
ELISA. The perturbation of mRNA levels corresponded with reduced
protein production. MM cells incubated with TGF-@1 or TGF-@32
antisense ODNs inhibited TGF-f31 and TGF-J32 secretion, respec
tively, as determined by ELISA (Fig. 2, C and D). This reduction in
TGF-@3production (between 40—75%of control values) was evident
over the 3 days of culture.
Effects of TGF4I Antisense ODNs in Vivo. The ability of TGF-(3
antisense ODNs to affect the growth characteristics of the tumor cells
that it was feasible
to investigate
the effects
of these
agents in vivo. Mice were inoculated i.p. with AC29 cells that were
previously either untransfected or transfected with either antisense
TGF-@3ODNs or a sense ODN. In the first experiments, mice receiv
mg TGF-@2 antisense ODN-transfected MM cells using phosphoro
3202
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INHIBITION
OF TUMOR GROWTH
A.
C,)
160
E
140
E
- -a-
E 120
.
- Control
TGF-132
.@100
@
80
0
@60
0
5
10
Time (days)
15
20
B.
C.)
E
E
1000
I
- -5-
0
800
0
600
E
TGF-1@2
- Control
L.
0
E 400
I@200
0
5
10
15
20
TIME (days)
25
30
Fig. 4. In vivo inhibition of the development of tumors by antisense TGFI32 ODNs. A,
mice were injected s.c. with either 2 X l0@AC29 MM cells transfected with a nonsense
ODNor AC29MMcellstransfectedwithTGF-f32ODNs(0.1 sg/2 X l0@cells).B,mice
were inoculated s.c. with 2 X l0@cells, and on days 1—5,mice received antisense TGF-f32
ODN(2 p@gIinjection)
i.p. Miceweremonitoredfor tumorgrowth,and tumorsize was
measured by microcalipers.
thioate-modified ODNs had significantly prolonged survival times
(50% survival at day 40 compared with 0% of mice receiving lipo
somes only at day 32; Fig. 3). Increased survival also corresponded
with a delay in the development of clinical symptoms.
We also investigated the effect of antisense TGF-(32 ODNs on the
development of s.c. tumors. AC29 cells transfected with antisense
TGF@32ODNs were inoculated s.c. into the flanks of recipient mice
and growth-monitored daily. This treatment delayed the tumor devel
opment from day 5 in the control group to day 11 in the experimental
group (Fig. 4A). However, once the tumor became palpable, the
kinetics of growth were similar in both groups. In a second type of
experiment, we investigated whether a repeated dose would further
delay or alter the kinetics of tumor growth. Mice were inoculated with
untransfected AC29 MM cells and then given multiple i.p. injections
of antisense ODNs. Fig. 4B shows that antisense TGF-j32 given for 5
days i.p. after s.c. inoculation of antisense-transfected AC29 cells
inhibited tumor development in a manner similar to that of the
previous experiment.
We further investigated the effect of delivering multiple doses of
TGF-j32 antisense ODNs by the administration of ODNs intratumor
BY ANTISENSE
TGF-f3
ally. Mice were inoculated s.c. with parental tumor and monitored
daily for tumor growth. Once the tumor became palpable at day 11,
intratumoral ODN treatment was commenced. Our results show that
TGF-f32 antisense ODNs significantly reduced the kinetics of tumor
growth in vivo, such that at the termination of the experiment (day 22),
the size of the treated tumors was only about 25% of that of the
control groups (P
0.0087; Fig. 5). Interestingly, we found no
difference between the efficacy of phosphorothioate ODNs and phos
phorothioate ODNs containing the C-S propyne analogues of uridine
and cytidine, this latter modification of the phosphorothioate deriva
tive having previously been reported to bind RNA with high affinity
and thus act as a more potent antisense inhibitor of protein translation
(29).
We next determined whether the intratumoral injection of TGF-/32
antisense ODNs acted by reducing the level of specific mRNA, as was
the case in the initial in vitro analysis. Tumors were excised from
animals at three time points (i.e. , days 1, 5, and 9 after the commence
ment of treatment), and mRNA was extracted. Using SQ-PCR to
determine the ratio of TGF-f32 mRNA to that of f3 actin 1 day after
commencement of the intratumoral injections, the relative level of
TGF-p2 mRNA was similar in both control and experimental groups,
whereas at 5 days, there was a substantial reduction in TGF-@2
mRNA in tumors treated with the antisense ODNs (Fig. 6). TGF-@32
mRNA increased again in the experimental group by day 9 of treat
ment, which corresponded to the time point at which there was an
increase in tumor growth.
To directly determine whether the in vivo effect of TGF-/3 antisense
ODN administration was to inhibit proliferation of tumor cells, BrdUrd
analysis was performed. Tumor-bearing animals were given an i.v. in
jection ofO.2 mg ofBrdUrd 16 h before sacrifice. Multiple tumor sections
sampled at various time points after tumor inoculation were stained using
specific monoclonal antibodies for the presence of BrdUrd, indicative of
DNA replication, and analyzed by confocal microscopy.
Confocal images showed that in the nonsense ODN-treated group,
proliferating cells (BrdUrd@) were present throughout the tumor (Fig.
7, A and B). In contrast, the antisense TGF-@32ODN-treated tumors
had significantly fewer BrdUrd@ cells between days 11 and 15 of
tumor growth (i.e. , days 1 and 5 after commencement of ODN
treatment; Fig. 7, C and D). This was particularly evident in the cells
in the center of the tumor, where only a few positive cells were
present. In the periphery of tumor mass of antisense-treated mice,
.
- -8-
U
E
TGF@2-T
- NO@SENSE
TGFB2-TC
--o--LlP@SOMES ONLY
0
E
,1+
0
>
5-
0
E
I-.
C
0
0
4,
—
0
10
12
14
16
18
20
22
Time (Days)
Fig. S. The effect of antisense TGF-j32 ODNs delivered intratumorally to established
tumors. Tumor-bearing mice were injected intratumorally with either TGF-f32-T ODNs
(antisense ODNs with the phosphorothioate backbone), TGF-@2-TC (phosphorothioate
ODNs containing the C-S propyne analogues of uridine and cytidine), nonsense ODNs, or
liposomes only. Results are reported as the mean of triplicate observations ± I SE;
P
0.0087
(two-tailed
Mann-Whitney
test).
3203
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@
,-
INHIB@ON OF TUMOR GROWTH BY ANTISENSE TGF-@
.
infiltrating
NONSENSE
D
TGF-@2
CD8@ or CD4@ T cells (data not shown). Overall, there
was no evidence that T-cell populations differed significantly between
the experimental and control groups. In addition, we did not observe
tumor-infiltrating natural killer cells in either the treated or control
groups. In all tumor groups, the predominate infiltrating leukocyte
was the macrophage, which was ubiquitous throughout the tumor.
There was also a smaller population of class Il-positive cells that did
not stain with macrophage markers by double-staining (data not
shown).
0
DISCUSSION
@
MM is an aggressive tumor that is refractive to conventional
therapies (30), having acquired a number of mechanisms that enhance
its capacity to survive and proliferate (11, 31—33).These mechanisms
11
15
19
include the production of several factors. An example of one of these
DAYS POST CHALLENGE
tumor-derived plunpotent factors is TGF-@3,which is a growth factor
Fig. 6. mRNA expression in tumors injected with either an antisense TGF@2 ODN or
for MM as well as a modulator of the immune response (8, 34, 35).
a nonsense ODN. Administration of ODNs intratumorally began on day 11 after tumor
inoculationandcontinueddailyuntiltheexperimentwasterminatedatday22.At 1,5,and We therefore proposed that the in vivo reduction of TGF-@ would
9 days after commencementof the treatment,mice were sacrificed,and mRNAwas have significant inhibitory effects on MM growth by: (a) reducing the
extractedfromexcisedtumors.mRNAspecificforTGF-I32wasdeterminedby SQ-PCR.
amount of growth factor available for the maintenance of tumor cell
This figure is representative of three separate experiments.
proliferation; and (b) allowing an effective antitumor immune re
sponse to be generated by reducing the effective concentration of the
there was a gradual increase in the number of proliferating cells, so immunomodulator.
that by day 19, there was little difference between the control and
We used antisense ODNs encapsulated in liposomes to target
experimental groups. This result correlates with the delayed tumor
TGF-@3mRNA and disrupt the production of protein. The advantage
growth observed in vivo and shows that antisense TGF-@2 ODNs
of antisense technology is that one can specifically target sequences of
injected intratumorally inhibit the proliferation of cells at the site of
interest within cells and effectively impair the function of the targeted
injection.
molecule by inhibiting the translation of the protein (17—19).Addi
To investigate the potential effect of TGF-(32 on the ilLs, fluores
tionally, the technology is permissive to gene therapy procedures and
cence-activated cell sorting and immunohistochemical analyses were
may be suitable in clinical trials.
performed. The proportion of T cells within the tumor did not alter
Our results show that production of TGF-j31 and -f32 is specifically
significantly in the TGF-@ antisense-treated group either as a percent
inhibited at both the level of mRNA and protein production and that
age of total cells within the tumor or as a percentage of cells with
this correlates with a reduced proliferative capacity in vitro. Signifi
lymphocyte forward and side light scatter characteristics. As the
cant reduction did not occur until day 3 of culture, presumably
tumor progressed, the number of CD8 cells declined. Examination of because the cells were able to use TGF-@3that had been produced
cryosections from tumors immunolabeled with a range of markers
before the ODN-disregulating translation. It is also clear that although
confirmed that tumors from animals treated intratumorally with either
the treatment reduces the amount of protein produced, its production
the nonsense ODN or antisense TGF-@32ODNs contained very few
is never totally suppressed. The data also show that either TGF-@1 or
@
a..
@
‘
‘
@
@
.
*,..* S..._
@
@
@
@
@
@
•.@
in mice receiving
intratumoral injections of antisense TGF-@2 ODNs.
.@ ,@
,..
.@_**
.
.
..
.
..
.
,-.-
.
..
.
.
*
*
.
,,I*,@. :.@
,,
*@,I.
—-
-,A•
@-:@ .
. - .
.
..
;-@•;@
@-
,‘...
.
*.I..'
‘@
,
‘.
mice were pulsed with BrdUrd 16 h
before sacrifice to allow the uptake of BrdUrd by pro
liferating cells. Multiple tumor sections sampled at var
ious points after tumor inoculation were stained for the
presence of BrdUrd and analyzed by confocal micros
copy. Tumor samples were from groups of mice that
received either nonsense ODNs (A and B) or antisense
:
•‘
@•
..
.
*;_•*@*
.
@\@@cAt.J:
,:
@‘
‘@:@‘
t@
e-.
‘.‘:
.,.@ ,4―
— P@
••‘I@,;
‘
-@
:@..
@.llt:
‘@
TGF-@2ODNs (C and D) intratumorally for 1 (A and
..;:@ni@'@. .@
C) and 5 (B and D) days.
.
I
@.
.
.“ • ,@.
.
4.•
@
@
.
:.
Fig. 7. Tumor cell proliferation
Tumor-bearing
@
@:
.
I..
@
@
‘@t't.,':
.
@
@
•
:
$
@
@
-.
“•*
..
@“
41H:;'.#•.
. .@ ,
,.,._
‘C.
.,@
@;_@_.
I
5..,@
:..
@@1@@sL―;?sa
,id,;*@t•*I
@.44
.
.‘
3204
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research.
lNHlB@ON OF TUMOR GROWTH BY ANTISENSE TGF-@3
-@2 is required for optimal growth in vitro, because the disruption of
either factor inhibits proliferation. It is not totally supnsing that there
is no differential effect, because both factors bind the same set of
receptors. In our model, we postulate that both factors must be
influencing the regulation of the cell cycle. TGF-@ is often cited as a
negative regulator of cell growth (36). It has also been shown that
these effects may be mediated by up-regulating cyclin-dependent
kinase inhibitor (37—39).Interestingly, other authors have demon
strated that some of these kinase inhibitors can be deleted or mutated
in MM (40—42).We are currently assessing the status of such inhib
itors in our model, although our results suggest that TGF-3 must also
be acting to directly stimulate proliferation. Our results showing that
either factor can be disrupted to obtain this effect cannot be explained
simply by the cross-inhibition of TGF-j3 production by the two types
of antisense ODNs, because both ODNs only inhibit their specific
protein mRNA. The obligatory requirement for either of the TGF-/3s
for tumor cell growth confirms that these factors are ideal targets for
in vivo therapy and suggests that only one isoform need be selected.
The initial in vivo results showed little difference in the ability of
the two isoforms to reduce tumor progression, but because we were
aiming to both reduce tumor growth and enhance the antitumor
immune response, a number of published observations led us to focus
on the @32isoform rather than the (31 isoform for the in vivo studies.
Experiments by two groups showed that TGF-@3lhas a positive effect
on T-cell proliferation and cytokine production (34, 43), whereas the
reduction of TGF-f32 in glioma models has been shown to prevent
immunosuppression of lymphocytes in vitro (44). We also have pre
liminary data that indicate that TGF-f32 rather than TGF-@3l sup
presses the ability of T cell receptor transgenic T cells to respond to
their target antigen in vitro.4 Thus, to enhance the immune response
while simultaneously inhibiting tumor growth, we used TGF-/32
ODNs in the in vivo experiments. The experiments reported herein
show that the administration of TGF-@32 antisense ODNs to mice
inoculated i.p. with MM significantly delayed the clinical symptoms
of tumor growth as well as increased their survival time. Preliminary
results using combined ASON for TGF-3l and TGF-j32 isotypes did
not provide an enhanced antitumor effect.5
Interestingly, the ODNs that were synthesized using phosphoro
thioate backbones were no more effective than the phosphodiester
ODNs, despite their increased in vivo survival (21, 45). One possible
explanation is that the effects of antisense TGF-3 ODNs occur within
the half-life of the natural nucleotides (half-life, approximately 20
mm), possibly because the ODNs were efficiently removed or de
graded in the peritoneal cavity. Therefore, increasing the effective
concentration of antisense ODN by using the more stable formulation
had no additional benefit.
In our work, we have found that sense sequence ODNs can exert
significant biological effects. A number ofeffects have been described
and include the destruction of the mRNA mediated by RNase H
recognition of the DNA:RNA hybrid (46). Other studies have shown
that RNase H, which cleaves mismatched RNA:DNA hybrids, can
inhibit protein synthesis when ODNs are targeted to the AUG initia
tion site, regardless of the rest of the ODN sequence (47). Other
nonspecific effects have been observed when two or more strings of
contiguous guanosines are present in the ODN sequence (48). In
addition, the mode of inhibition is strongly dependent on the chem
istry of the ODN and its length
(20).
The ODNs were thus designed to minimize these potential conse
quences (Table 2), although we still observed some inhibitory results
using sense controls. The mechanisms by which this occurred are not
4 A.
Marzo,
manuscript
in preparation.
5 A.
Marzo,
unpublished
observations.
clear, because these effects were specific in some respects, because
@-actinmRNA was unaffected in the in vitro assays, and sense
TGF-/3l ODNs only affected TGF-f3l and not TGF-f32 and vice versa,
illustrating the need for empirical testing of control ODNs. Scrambled
sequences
still represent
good
controls
because
they did not show
nonspecific inhibitory effects in these experiments. It has also been
recently reported that the dinucleotide sequence CpG, which is present
in the TGF4J
antisense
ODNs,
is stimulatory
for both T and B cells
(49, 50). Although this is a potential problem in interpreting our data,
the fact that all control ODNs also contain this motif, as well as the
lack of any evidence for an increased immune response, probably
negates this factor.
Although the stable transfection of antisense DNA has been re
ported to inhibit the growth of MM ( 13), stable transfection is not
readily applicable as a therapy for solid tumors. The use of liposome
encoated antisense ODNs, on the other hand, is adaptable to this type
of therapy. Previously reported work investigating the efficacy of
antisense ODNs administered at the time of tumor inoculation or
shortly afterward demonstrated in nude mice that continuous infusion
of antisense ODNs at the time of tumor inoculation could result in up
to a 50% reduction in tumor mass (51, 52). Another group, which
pretreated tumor cells with TGF-/3l antisense ODNs before tumor cell
inoculation, was able to reduce the metastatic capacity of the murine
fibrosarcoma line (53). Recently, it has been shown that antisense
ODNs active against the parathyroid hormone-related peptide can
reduce the growth and metastasis of a pituitary tumor for up to a week
after tumor inoculation in a rat model (54). Our results show that
liposome-encapsulated TGF-j3 antisense ODNs are capable of reduc
ing the tumorigenicity of MM. In addition, we go one step further,
because we have investigated the effect of antisense TGF-j32 ODNs
delivered intratumorally to an already palpable tumor, an approach
that is more relevant to the treatment of established disease. The data
show that tumor development could be significantly delayed. As far as
we are aware, this is one of the few demonstrations of antisense ODN
inhibition of the growth of an established tumor in immunocompetent
mice. Because TGF-j3 has the potential to increase angiogenesis and
thus provide support for tumor growth, the reduction in tumorigenesis
after TGF-@ antisense ODN treatment could be ascribed to decreased
vasculature (55, 56). Although this possibility cannot be ruled out, the
in vitro studies
clearly
show that TGF-f3 is a growth
factor for MM
cells, and ASON treatment can be directly attributed to reduction in
growth factor activity. The reduced growth of antisense ODN-treated
tumors was confirmed using BrdUrd incorporation and confocal mi
croscopic analysis that showed little tumor proliferation at the center
of the tumor (i.e. , at the site of injection), whereas tumor cells
continued to proliferate at the periphery of the tumor mass, where
there was presumably a much lower concentration of antisense ODNs.
We interpret this result to mean that the liposome-encapsulated ODNs
are only effective over a relatively short distance and that as the tumor
gets slowly but progressively larger, the efficacy wanes. Thus, the
ability of antisense ODNs to adequately reduce solid tumor growth
will be reliant on the development of efficient delivery systems.
In the TGF-@32antisense ODN-treated mice, a small increase in the
number of ilLs was noted. This was also observed in previous stable
transfection experiments (13). We usually find that TILs are predom
inantly located at the periphery of the tumor mass rather than scattered
through the stroma, implying that their ability to penetrate the tumor
mass
is limited
(25).
This
is consistent
with
in vitro
studies
that
showed that TGF-@32can inhibit lymphocyte migration and may be
one of the factors that prevents lymphocytes from invading the blood
brain barrier (57). If TGF-@ is also reducing lymphocyte migration in
our tumor model, the ability of antisense ODNs to decrease local
TGF-f32 production could explain the transitory increase in TIL
3205
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research.
INHIBITION
OF TUMOR
GROWTH
numbers that lasted only as long as peripheral tumor growth was
restricted (i.e., until day 19 of tumor growth). We did not find any
other evidence that the reduction of TGF-f32 invoked a more potent
antitumor response. One explanation for this lack of immune poten
tiation by antisense TGF-@ ODNs may be that the amount of protein,
although reduced enough to slow tumor growth, was never suffi
ciently diminished to avoid TGF-/3 modulation of the immune system.
Thus, although TGF-@ seemed to be the perfect candidate due to its
dual properties of tumor growth factor and immune modulator, it is
probably naive to imagine that targeting one molecule will induce the
complete reversal of tumorigenicity. It is possible that combination
therapies may be more effective at reducing tumor growth, such as
combining antisense TGF-j3 ODNs with more potent immunostimu
latory molecules such as interleukin 2 or interleukin 12. Additional
studies are being undertaken to test this suggestion. Overall, our
results have implications for immunotherapies of other solid tumors as
well as for understanding how TGF-(3 may be involved in attenuating
the immune response.
The data presented in this paper provide an encouraging basis for
additional investigations into the therapeutic potential of antisense
ODN technology, particularly for the treatment of solid tumors such
as MM.
BY
ANTISENSE
TGF-@3
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ACKNOWLEDGMENTS
Many thanks to Dr. Richard Lake for thorough review of the manuscript and
valuable comments. We also acknowledge the technical assistance of Drs. N.
Bilyk and U. Seydel with confocal microscopy.
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Antisense Oligonucleotides Specific for Transforming Growth
Factor β2 Inhibit the Growth of Malignant Mesothelioma Both in
Vitro and in Vivo
Amanda L. Marzo, David R. Fitzpatrick, Bruce W. S. Robinson, et al.
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