[Cancer Research 60, 1797-1799, April 1, 2000]
© 2000 American Association for Cancer Research
Gene Transfer into Brain Parenchyma Elicits Antitumor Effects1
Hassan M. Fathallah-Shaykh2,
Abdallah I. Kafrouni,
Li-Juan Zhao,
George M. Smith3 and
James Forman
Departments of Neurology [H. M. F-S., A. I. K., L-J. Z.], Anesthesia and Pain Management [G. M. S.], and Center For Immunology [J. F.], The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235
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ABSTRACT
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Gene therapy strategies for cancer currently aim at targeting gene
delivery to the malignant cell. In a mouse model of intracerebral Lewis
lung carcinoma (3LL), adenoviral vectors transduce not only 3LL cells
but also brain parenchymal cells including endothelial cells, neurons,
microglia, and astrocytes
in vivo. Furthermore,
transgene expression persists longer in brain than in tumor. Transfer
of IFN-
into brain parenchymal cells rather than tumor is both
necessary and sufficient to generate antitumor therapeutic benefits.
Therefore, parenchymal cells represent an effective and necessary
target for delivery of genes that render the brain uninhabitable by the
tumor.
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Introduction
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Current experimental gene therapy strategies for malignant brain
tumors aim at targeting delivery of the transgene to the tumor cell.
The genes transferred include those that induce a suicide effect,
modulate the immune response, arrest the cell cycle, induce apoptosis,
or inhibit neovascularization (1, 2, 3, 4, 5, 6, 7, 8, 9, 10)
. Historically,
delivering the suicide gene herpes simplex thymidine kinase into brain
tumors was the first gene therapy strategy for brain cancer to show
efficacy in animal models, and this therapy has been tested in humans
(2
, 11)
. Because of the inability of retroviral vectors to
infect quiescent cells, Culver et al. (2)
hypothesized that recombinant retroviruses can target suicide gene
delivery into rapidly multiplying tumor cells while sparing the
background of nondividing neural tissue. Data from this laboratory
showed that treatment of mice harboring a poorly immunogenic carcinoma
(3LL) implanted in the brain with an adenoviral vector to deliver
IFN- (AdIFN) at the same coordinates as the tumor elicits
significant prolongation of survival times and tumor rejection. In this
mouse brain tumor model, the antitumor effects of AdIFN are independent
of adaptive cellular immune mechanisms and appear to be mediated by
antiangiogenesis (12)
. Evidence against a cellular
immune-mediated response include: (a) absence of a memory
immune response on challenge; (b) lack of antitumor effects
at sites distal to inoculation of AdIFN; and (c)
preservation of the therapeutic benefits in scid, beige,
iNOS C57/BL6 knockout mice and mice treated with
NG-nitro-L-arginine-methyl ester. High
concentrations of IFN- do not inhibit tumor growth in
vitro, making it unlikely that the antitumor effect of this
treatment acts directly on the growth of the tumor cell. However, gene
transfer of IFN- inhibits neovascularization of the tumor in the
avascular s.c. space in a Matrigel assay in vivo, and AdIFN
induces apoptosis of endothelial cells in vivo, thus
supporting the idea that AdIFN represses tumor growth by inhibiting
angiogenesis (12)
.
The experiments in this study were designed to determine:
(a) the identity of the brain parenchymal cells transduced
with AdIFN; and (b) whether the antitumor effects are
generated by delivery of IFN- into the tumor cell versus
brain parenchyma.
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Materials and Methods
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Animals and Vectors.
C57/BL6 mice were purchased from the Jackson Laboratories (Bar Harbor,
ME). Animal care was in accordance with institutional guidelines. 3LL
(Lewis lung carcinoma; C57/BL6) is a generous gift from Dr. I. Fidler
(M. D. Anderson Cancer Center, Houston, TX). The generation of
adenoviral vectors AdIFN and AdBGAL was described elsewhere
(12)
. The titers of AdIFN and AdBGAL of viral
particle:plaque-forming unit ratios are 43 and 81, respectively. Mice
were anesthetized and injected intracerebrally as described previously
(12)
. Statistical calculations were performed by the JMP
software (SAS Institute, Cary, NC).
Tissue Staining.
Antibodies against mouse IFN- (American Type Culture Collection,
Manassas, VA) and tomato lectin (Sigma Chemical Co., St. Louis, MO),
anti-GFAP (DAKO, Carpenteria, CA), anti-NeuN (Chemicon, Temecula, CA),
and anti-factor VIII (DAKO) were used. Biotinylation reactions were
performed following the manufacturer’s specifications (Vector
Laboratories, Burlingame, CA). Immunofluorescence and
immunohistochemistry were performed as described previously
(12)
.
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Results and Discussion
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To identify the cell types transduced with AdIFN in
vivo, groups of mice that received intracerebrally 3LL implants
were injected 10 days later with AdIFN at the same coordinates as the
tumor, and their brains were sectioned 4 days later. Immunofluorescence
staining for astrocytes (green) and IFN- (red)
reveals IFN- reactivity predominantly in the brain, outside and
surrounding the area of the tumor (Fig. 1
). Furthermore, double immunofluorescence staining demonstrates that
astrocytes (Fig. 2,
a–c
), microglia (Fig. 2, d–f
), neurons (Fig. 2, g–i
),
and endothelial cells (Fig. 2, j–l
) are all transduced with
AdIFN in vivo.
To examine whether selective gene transfer into brain parenchymal cells
is sufficient to generate the antitumor effects of AdIFN, mice first
received implants of either AdIFN or AdBGAL and were then reinjected 4
days later with
wt4
3LL at the same coordinates and followed for survival. The therapeutic
benefits of AdIFN in these experiments were identical to the effects of
AdIFN in the treatment of mice with 3LL brain tumors (Fig. 3, a and b
); AdIFN generated statistically significant prolongation of survival
times as well as tumor rejection in 4 of 10 mice (Fig. 3b
).
The latter survived for >85 days, and histological analysis of their
brains showed cavity formation (data not shown). To study the
biological effects of selective gene transfer of IFN- into tumor
cells, 3LL cells transduced in vitro to secrete 1.125 µg
IFN-/106 cells/24 h were implanted in mice, and the mice
were followed for survival. The survival benefits in these experiments
were modest but statistically significant; however, none of these mice
survived longer than 36 days (Fig. 3c
).
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Fig. 3. Gene transfer into brain parenchymal cells but not tumor
cell reproduces the therapeutic effects of AdIFN on 3LL brain tumors.
Mice received intracerebral implants of 1500 3LL cells in 3 µl. Two
days later, mice were treated with either AdIFN (10 µl; 24 x 109 viral particles; n = 8) or AdBGAL (10 µl; 35 x 109 viral
particles; n = 6) at the same coordinates
(a). Survival times were examined by Kaplan-Meier analysis.
Mean survival times were 29.7 days and >57.1 days for AdBGAL- and
AdIFN-treated mice, respectively (log-rank P < 0.0003). Animals were also injected with either AdIFN or AdBGAL
(10 µl; 24 x 109 viral particles;
n = 10 each) first, followed 4 days later by
3LL at the same coordinates (b). Mean survival times were
31.6 days and >54.6 days for AdBGAL- and AdIFN-treated mice,
respectively (log-rank P < 0.0093).
c shows the survival times of animals that received
intracerebral implants of 3LL cells transduced in vitro with
either AdIFN or AdBGAL (n = 10 each). One
million 3LL cells were cultured in the presence of AdIFN or AdBGAL
(45 x 1010 viral particles each) in
vitro. Two days later, the cells were washed extensively with PBS,
and 1500 cells were injected intracerebrally. Mean survival times were
24.1 and 31.2 days for AdBGAL- and AdIFN-treated mice, respectively
(log-rank P = 0.0002).
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To determine the persistence of transgene expression in brain and
tumor, groups of mice (n = 6 each)
received implants of: (a) 3LL cells transduced with AdBGAL
in vitro; or (b) AdBGAL followed 4 days later
with wt 3LL cells at the same coordinates. AdBGAL-transduced tumor
cells instead of AdIFN transduced tumor cells were used because IFN-
secreted by the tumor induces microglia to secrete endogenous IFN-,
thus obscuring the localization of the virus. The brains were sectioned
10 days after the first injection. Although all 3LL cells transduced
with AdBGAL in vitro contained ß-gal activity at day 0
(Fig. 4a
), none of the brains implanted with these tumors stained for ß-gal at
day 10 (Fig. 4, b and c
). Nonetheless, mice first
injected with AdBGAL showed prominent ß-gal expression in brain
parenchyma surrounding the tumor (Fig. 4, d and e
).
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Fig. 4. Transgene expression persists longer in brain than tumor.
One million 3LL cells were transduced with AdBGAL in vitro
(45 x 1010 viral particles); 2 days later,
they were stained for ß-gal (a) or washed extensively, and
1500 cells were injected intracerebrally into naïve mice
(n = 6). Ten days after injection,
consecutive frozen sections of the brain were stained for ß-gal
activity and reacted with anti-GFAP antibodies (b) or
stained with H&E (C). Arrows point to the tumor
site, showing a paucity of astrocytes (b and c).
Wheraes all transduced 3LL cells expressed ß-gal before injection
into the brain (a), intracerbral tumors examined 10 days
after implantation do not show ß-gal activity (b and
c). C57/BL6 mice (n = 6) were
implanted with AdBGAL followed 4 days later with 1500 wt 3LL cells
(d and e). Ten days after the viral injections,
consecutive frozen sections of the brain were stained for ß-gal
activity and reacted with biotinylated anti-GFAP antibodies
(d) or stained with H&E (e). ß-Gal
expression is prominent in brain parenchyma surrounding the area of the
tumor showing a paucity of astrocytes (d and e,
arrows point to ß-gal staining. o.m. is x200 for
a–e.
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The results show that AdIFN transduces not only tumor cells but also
brain parenchymal cells including endothelial cells, microglia,
neurons, and astrocytes in vivo. Although implantation of
3LL cells transduced in vitro into the brain is associated
with modest survival benefits (Fig. 3c
), it fails to
duplicate the therapeutic effects of AdIFN in treating 3LL brain tumors
(Fig. 3a
), making it unlikely that the antitumor effects of
AdIFN in the latter model are mediated solely by transduction of the
tumor cell. Thus, the data argue that gene transfer into brain
parenchyma is necessary to optimize the therapeutic benefits.
Furthermore, selective transfer of IFN- into brain parenchyma
reproduces the survival benefits of AdIFN in treating 3LL brain tumors
(Fig. 3b
), suggesting that brain transduction alone is
sufficient to generate the therapeutic response (Fig. 3a
).
The gains of gene transfer into brain parenchymal cells may stem from
the fact that unlike rapidly multiplying tumor cells, they are less
likely to lose the transgene and are more likely to produce sustained
high amounts of the gene product for longer periods of time (Fig. 4
).
Targeting gene transfer into brain parenchymal cells located in
proximity of the tumor is an attractive adenoviral-mediated strategy
for delivering molecules that are either antiangiogenic, exert
immunomodulatory effects on both the tumor and immune cells, inhibit
tumor cell growth directly, or induce selective tumor cell apoptosis.
Tumor cells constitute an unstable source for transgene production
because they multiply rapidly, thus diluting out the episomal transgene
(Fig. 4
). Furthermore, by killing a large number of malignant cells, a
potentially successful therapeutic strategy may fail because of
prematurely reducing or eliminating transgene production by the tumor.
Because brain parenchymal cells are nondividing or replicate slowly,
they make up a reliable and potentially controllable factory for
producing a gene product cloned under an inducible promoter (Fig. 4
).
Furthermore, such a strategy is particularly attractive for gliomas
because they tend to recur within a few centimeters of the original
tumor site. In summary, the results present a proof of principle that
the tumor bed, in this instance, brain parenchymal cells, is an
effective and necessary target for delivering genes that render the
brain uninhabitable by the tumor.
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Acknowledgments
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We are indebted to Roger Rosenberg for support and helpful
discussions.
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FOOTNOTES
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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.
1 Supported by NIH Grants R01-CA81367 and
R29-CA78825 and by the 1998 Scholar Award from the Children’s Brain
Tumor Foundation of The Southwest. 
2 To whom requests for reprints should be
addressed, at The University of Texas Southwestern Medical Center at
Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9036. Phone: (214)
648-9413; Fax: (214) 648-7992; E-mail: hassan_fathallah{at}hotmail.com
3 Present address: University of Kentucky,
Department of Physiology, Albert B. Chandler Medical Center, 800 Rose
Street, Lexington, KY 40536-0298.
4 The abbreviations used are: wt, wild-type;
ß-gal, ß-galactosidase; o.m., original magnification; GFAP, glial
fibrillary acidic protein.
Received 12/15/99.
Accepted 2/16/00.
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ABSTRACT
Introduction
