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Experimental Therapeutics |
UMR1582 CNRS-IGR-Rhône-Poulenc [A. B., P. O., P. Y., M. P.] and Service de Physique [M. R.], Département de Biologie Clinique [J-M. B.], and Service de Médecine Nucléaire [M. S.], Institut Gustave Roussy [M. S.], 94805 Villejuif, France, and Dipartimento di Medicina Sperimentale e Clinica, Università di Catnzaro, 88100 Catanzaro, Italy [S. F.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Iodide accumulation in the thyroid gland is ensured by the NIS,3 a transmembrane glycoprotein present in the basolateral pole of thyroid follicular cells. NIS-mediated iodide uptake is an active transport process that occurs against the electrochemical gradient in I- anions and is competitively inhibited by thiocyanate and perchlorate anions (7 , 8) . The cDNAs of the rat and human NIS genes have been cloned recently and code for proteins of 618 and 643 amino acids, respectively (9 , 10) . The two proteins are 84% identical and have been predicted to be integral membrane proteins displaying 12 (9, 10, 11) or 13 membrane-spanning domains (12) . NIS expression is not strictly limited to the thyroid but also occurs in several extrathyroidal tissues, including the salivary glands, the gastric mucosa, and the mammary gland (13) . In these tissues, however, iodide is not organified (13) .
The cloning of the NIS gene constitutes an important step toward the understanding of the molecular mechanisms underlying iodide transport abnormalities in thyroid pathologies. Indeed, several cases of hypothyroidism with low iodide uptake were linked to inactivating mutations in the NIS gene (14 , 15) . Similarly, the low or absent iodide transport observed in thyroid cancer tissues was correlated with a low or absent NIS expression (6 , 16 , 17) . Importantly, NIS-mediated iodide transport does not require the follicular organization of the thyroid, as demonstrated in vitro in bovine (18) , porcine (19) , human (20) , and rat thyroid cells (7) . Several in vitro studies also showed that transfer of the NIS gene into nonthyroid cells, either by transfection of NIS cDNA (9 , 10 , 21 , 22) or with a retroviral vector (23) , led to iodide uptake by the transduced cells. Coupling delivery of the NIS gene into tumor cells with 131I administration may therefore open new avenues to treat cancer.
Adenoviral vectors are particularly well suited for cancer gene therapy. They lead to a transient but robust expression of the transgene, and efficient in vivo gene transfer has been reported in numerous tissues, including the thyroid (24, 25, 26) . In addition, concentrated adenovirus preparations can be obtained, which constitutes a clear advantage over other viral vectors such as retroviruses for an optimal in vivo gene transfer. The commonly used adenoviral vectors are extremely attenuated and lack at least the viral early transcription regions E1 (essential for replication) and E3. Such vectors still exhibit some level of cytotoxicity, which can be viewed as an advantage for destructive strategies such as cancer gene therapy (for review, see Ref. 27 ).
The aim of this work is to demonstrate the feasibility of using an adenoviral vector to deliver the NIS gene into human tumors, with the goal of enabling them to concentrate radioactive iodine. For this purpose, we constructed and characterized a recombinant adenovirus expressing the rat NIS gene (AdNIS) as a first step toward a targeted radiotherapy of tumors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E1–E3 recombinant adenovirus expressing the rat
NIS gene under the control of the immediate early promoter
of the cytomegalovirus (pCMV). The rat NIS gene
[nucleotides 74–2046 of the published sequence (9)
] was
cloned as a AatII (blunt-ended by Mung Bean nuclease
treatment)-HindIII fragment in the
PvuII-HindIII sites of the pCEP-4 vector
(Invitrogen) to add a promoter (pCMV) and a polyA (SV40 polyA).
The obtained plasmid was called pAB1. A 3711-bp
SspI-EcoRV fragment of pAB1 was then cloned in
the shuttle vector pXL3048 linearized by EcoRV. pXL3048 is a
KmR-SacB-ColE1 derivative (28)
containing the left end of the Ad5 genome (nucleotides 1–386), a
polylinker with three unique cloning sites (EcoRV,
BamHI, and SalI), and part of the Ad5
pIX gene (nucleotides 3446–4296). The shuttle vector
containing the NIS expression cassette was called pAB2. The recombinant
adenoviral genome encoding NIS was obtained by homologous recombination
between plasmids pAB2 and pXL3215 in Escherichia coli, as
described previously (28)
. pXL3215 contains the Ad5 genome
bordered by two PacI sites and carrying deletions within E1
(nucleotides 386–3446) and E3 (nucleotides 28592–30470). After
recombinational cloning in E. coli, the adenoviral genome
was excised by PacI digestion, and the AdNIS virus was
recovered by transfecting 10 µg of PacI-digested DNA into
293 cells by the Lipofectamine-based procedure (Life Technologies,
Inc.).
Control Adenoviral Vectors.
Recombinant adenoviruses expressing no transgene (AdCO1) or encoding
ß-galactosidase (Adßgal) were used as negative controls in this
study and have been described previously (29
, 30) .
Virus Amplification, Purification, and Titration.
All viral stocks were prepared from infected 293 cells
(31)
by standard procedures (30)
. After a
two-step purification on CsCl gradients, viral stocks were desalted by
using Pharmacia G50 columns (Orsay, France) and frozen at
-80°C in PBS containing 7% glycerol. Viral titers were calculated
by dilution plaquing onto 911 cells (32)
and expressed in
PFU/ml.
Cell Lines.
Unless stated otherwise, all cell culture media and reagents were
purchased from Life Technologies, Inc. Rat thyroid FRTL-5 cells (ATCC
CRL-8305) were routinely grown in Coon’s modified Ham’s F12 medium
(Sigma) supplemented with 5% donor calf serum (Life Technologies, Inc.
16030) and 10 µg/ml insulin (Sigma I 1882), 10 nM
hydrocortisone (Sigma H 0396), 5 µg/ml transferrin (Sigma T 1147), 10
ng/ml somatostatin (Sigma S 1763), 10 ng/ml
glycyl-L-histidyl-L-lysine acetate (Sigma G
7387), and 10 milliunits/ml thyrotropin (Sigma T 8931). SiHa (ATCC
HTB-35), MCF7 (ATCC HTB-22), T-47D (ATCC HTB-133), DU 145 (ATCC
HTB-81), and HT-29 (ATCC HTB-38) cells were maintained in DMEM
supplemented with 10% heat-inactivated FBS. PC-3 cells (ATCC CRL-1435)
were grown in F-12K Nutrient Mixture (Kaighn’s modification)
supplemented with 10% heat-inactivated FBS. A549 cells (ATCC CLL-185)
were maintained in minimum Eagle’s medium supplemented with 10%
heat-inactivated FBS and 1% nonessential amino acids. 293 and 911
cells were grown in minimum Eagle’s medium supplemented with 10% FBS
and 1% nonessential amino acids and DMEM supplemented with 10% FBS,
respectively.
Anti-NIS Antibodies.
A peptide spanning the COOH-terminal (600–618) region of rNIS was
synthesized by a conventional solid-phase method using an Applied
Biosystems Model 431A peptide synthesizer. The identity and purity of
the (600–618) peptide were verified by amino acid analysis and peptide
microsequencing. The synthetic peptide was conjugated to keyhole limpet
hemocyanin using benzidine as the coupling agent on the
Lys600 residue. Two rabbits were immunized by intradermal
injection of the synthetic peptide-carrier conjugate. After two boosts
at 3-week intervals, animals were bled, and their sera were tested in
an ELISA. Antisera, at various dilutions, were verified for their
capacity to react with the rNIS synthetic peptide coated on microtiter
plates. Antibody binding was then revealed by peroxidase-labeled goat
antirabbit antibody (Nordic, Tilburg, the Netherlands).
Immunofluorescence.
SiHa cells were seeded in 4-wells SonicSeal Slides (Nunc, Inc.) at a
density of 4 x 105 cells/well. The next
day, cells were infected with virus-containing culture supernatant from
the first viral amplification step; a 5-fold dilution was used, and
infection was carried out for 1 h in 200 µl of medium before the
addition of 800 µl of medium. Twenty-four h after infection, cells
were fixed for 15 min in PBS-4% formaldehyde, washed twice with PBS,
and permeabilized with PBS-0.2% Triton X-100 for 10 min. After two
other washes with PBS, cells were incubated overnight at 4°C in
PBS-0.5% FBS. The NIS protein was detected using the rabbit polyclonal
antibodies described above; cells were incubated for 90 min with the
anti-NIS antibody diluted 1:500 in PBS, washed three times with
PBS-0.5% FBS, and then incubated for 30 min with a
fluorescein-conjugated secondary antibody (Vector Laboratories, Inc.,
Burlingame, CA). After further washing in PBS-0.5% FBS, slides were
mounted and observed with a fluorescence microscope.
Infection Conditions for Iodide Uptake Experiments.
Cells were seeded in 24-well plates 3 days before the experiment to
achieve between 5 x 105 and 106
cells/well at the day of infection. Cell numbers were determined
immediately before infection as the average cell content of two wells.
Cells were infected at the indicated MOI in 200 µl of medium for
1 h, and then 800 µl of medium were added in each well. For each
cell line, the medium used for infection was the same as the culture
medium.
In Vitro 125I Uptake Experiments.
Iodide uptake experiments were performed 28–30 h after virus
infection, using the method of Weiss et al.
(7)
. Briefly, cells were washed once with 1 ml of HBSS
buffered to pH 7–7.5 with 10 mM HEPES (bHBSS). Iodide
uptake was then initiated by adding 0.5 ml of bHBSS containing 0.1
µCi of 125I per well. After the indicated time of contact
with iodide, cells were washed once with ice-cold bHBSS and incubated
for 20 min in 1 ml of ice-cold ethanol. The ethanol was then recovered,
and radioactivity was quantified (cpm) with a well gamma-counter
(Beckman gamma 5500 B).
In Vitro Cell Killing with 131I and
Clonogenic Assay.
Cells were seeded in 24-well plates and infected at a MOI of 10, as
described above. Twenty-five h after infection, cells were washed once
with 0.5 ml of bHBSS and incubated with 0.5 ml of bHBSS (control) or
0.5 ml of bHBSS containing 10 µCi of 131I. After 5 h
of contact with 131I, cells were washed twice with bHBSS,
trypsinized, and counted. For each condition [noninfected cells,
AdNISinfected cells, and cells infected with a control adenovirus
(AdCO1)], cells were plated in triplicate in 6-well plates (1000
cells/well) and incubated for 1 week at 37°C. Cells were then washed
once with PBS and stained with a cristal violet solution (for 250
ml, 0.5 g of cristal violet, 25 ml of 40% formaldehyde, 50
ml of ethanol, and 175 ml of water). Colonies of more than 30 cells
were counted, and the means and SD were determined for each condition.
Results are expressed as the percentage of surviving cells,
i.e. the percentage of colonies obtained after treatment
with 131I compared to treatment with bHBSS alone, and are
representative of two separate experiments.
Tumor Induction and in Vivo Iodide Uptake
Experiments.
Female nude mice (6–8 weeks of age) were irradiated (5
Gy) the day before injection of the tumor cells. Tumors were induced by
s.c. injection of 200 µl of sterile PBS containing 5 x 106 SiHa or MCF7 cells. In the case of MCF7 tumors,
the cell suspension contained 50% Matrigel (Becton Dickinson). When
tumors had reached 5–8 mm in diameter (approximatively 3 weeks after
cell injection), a 10-day thyroxine treatment was initiated to suppress
thyroid iodine uptake; each day, animals were injected i.p. with 2 µg
of L-thyroxine (Roche) diluted in 100 µl of PBS. Seven
days after the onset of the L-thyroxine treatment, the
AdNIS virus was injected into the tumors (2 x 109 PFU in 100 µl of PBS), and iodide uptake was assessed
3 days later. For kinetics and quantitative uptake experiments, 6 µCi
of 125I were injected i.p. in 200 µl of sterile PBS. The
presence of radioactive iodide in the tumors was recorded up to 400 min
after the injection of radioactive iodide, using a small
radiation-sensitive probe (Europrobe-Eurorad, Strasbourg, France). For
quantitative analysis of the amount of 125I present in the
tumors, mice were sacrificed 90 min after the injection of radioactive
iodide, the weight of the tumors was determined, and radioactivity was
quantified using a calibrated well gamma-counter (Compugamma 1282; LKB)
for 1 min. For experiments with 131I, mice were injected
i.p. with 30, 60, or 90 µCi of 131I in 200 µl of
sterile PBS (corresponding in terms of radioactivity/weight to doses
generally used in human therapeutics); five animals were included in
each group, and tumor sizes were followed for 2 weeks. For imaging
experiments, 50 µCi of 123I were injected i.p. in 200
µl of sterile PBS; 90 min later, animals were anesthetized, and an
image was taken (gamma camera DHD, SMV, BUC France). In all
cases, the tumors were removed and analyzed by immunohistology.
Immunohistology.
Removed tumors were fixed in 5% acetic acid, 75% absolute ethyl
alcohol, 2% of formalin (40%), and 18% water for paraffin block
preparations. To examine the histological aspect of the tumor, paraffin
sections (5-µm thick) were stained with H&E-saffron. The presence of
the NIS protein was revealed by incubating the sections for 1 h
with anti-NIS polyclonal antibodies (see above) at a 1:1300 dilution
and then incubating sections for 30 min with a goat antirabbit
secondary antibody conjugated to peroxidase (Envision; Dako).
3-Amino-9-ethylcarbazole (Envision kit; Dako) was used to reveal the
markers, and sections were counterstained with Mayer’s hematoxylin
(1:2).
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RESULTS |
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shows that AdNIS-infected cells were clearly labeled and that the NIS
protein was localized at the cell surface. On the contrary, noninfected
SiHa cells were not labeled (Fig. 1B)
. In addition, cells
infected with a control adenovirus (Adßgal) and AdNIS-infected cells
treated with PBS in place of the primary antibody did not display any
staining (data not shown), confirming that the observed signal was
linked to the expression of the NIS protein. The AdNIS vector thus
leads to high-level expression and correct localization of the NIS
protein within the infected cells.
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. Surprisingly, at a higher MOI, a lower amount of iodide was taken up
(Fig. 2A)
; possible explanations include a direct
cytotoxicity of the adenoviral vector or a toxic effect of NIS
overexpression in the cell membranes. In addition, cells infected with
a control adenovirus (Adßgal) did not accumulate more iodide than
noninfected cells (data not shown), demonstrating that iodide uptake
was specifically linked to expression of the NIS protein. Taken
together, these results demonstrate that the AdNIS virus drives
expression of a functional protein in vitro and leads to
efficient iodide uptake after infection.
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. In FRTL-5 cells, iodide uptake was already inhibited more
than 90% in the presence of 30 µM NaClO4, as
described previously (7
, 23)
. Likewise, iodide uptake in
cells infected by AdNIS (MOI 10) was inhibited by NaClO4 in
a dose-dependent manner. However, results were quite different from
those obtained for the thyroid cells: at an inhibitor concentration of
30 µM, the process was only inhibited to 54%, and a
concentration of 300 µM was required to inhibit uptake by
more than 90%. A probable explanation for this observation is that
SiHa cells are easily infected by adenoviruses and that AdNIS-infected
cells thus probably express very high levels of NIS.
We then studied the kinetics of iodide uptake in AdNIS-infected SiHa
cells and compared it with that of FRTL-5 cells (Fig. 2C)
.
Equal numbers of FRTL-5 cells, noninfected SiHa cells, and
AdNIS-infected SiHa cells (MOI 10) were incubated for 5–120 min
with 0.1 µCi of 125I, and the amount of incorporated
iodide was quantified at several time points. As expected, noninfected
SiHa cells did not accumulate iodide, even after a 2-h incubation with
125I. Iodide uptake was very rapid in FRTL-5 cells, as
described previously (7)
. In the case of AdNIS-infected
cells, the initial kinetics of iodide uptake was similar to that
observed for FRTL-5 cells, with a maximal level reached after 30 min.
However, for longer incubation times, the amount of iodide retained in
the cells decreased significantly, suggesting an efflux effect.
Interestingly, AdNIS-infected cells accumulated three to five times
more iodide than the FRTL-5 cells. Transfer of the NIS gene
by the AdNIS vector thus leads to rapid and perchlorate-sensitive
iodide uptake, as described for cells naturally expressing a functional
NIS protein.
The AdNIS Vector Is Functional in Various Human Tumor Cell Lines.
Iodide uptake experiments were performed on several human tumor cell
lines, namely MCF7 and T-47D (mammary gland), PC-3 and DU 145
(prostate), A549 (lung), and HT-29 (colon) cells. For each cell line,
MOIs of 10 and 25 were tested, and iodide uptake was measured after 15
min of contact with 0.1 µCi of 125I. For all cell lines,
AdNIS infection at a MOI of 10 led to a highly significant iodide
uptake. The amount of iodide taken up varied slightly from one cell
line to the other, being increased 35–225 fold as compared with
noninfected cells (Fig. 3)
. These differences are partially due to small variations in cell numbers
(see Fig. 3
) but also reflect the capacity of the adenoviral vector to
infect the different cell lines. Increasing the MOI did not have the
same effect on all the cell lines tested. At a MOI of 25, MCF7, PC-3,
A549, and HT-29 cells displayed an increased capacity to concentrate
iodide, whereas almost no difference was observed for T-47D cells; on
the contrary, the capacity of DU 145 cells to take up iodide decreased,
similar to what we observed for SiHa cells (see Figs. 2
and 3
). These
differences probably reflect the more or less pronounced cytotoxic
effect of the adenoviral vector on the various cell lines tested.
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, iodide uptake was again inhibited in a dose-dependent
manner by perchlorate anions. However, in contrast to what was observed
with AdNIS-infected SiHa cells, a concentration of 30 µM
was sufficient to obtain an inhibition of more than 95%. This
difference again reflects the capacity of the viral vector to infect
the different cell lines, which is at least 10–20 times higher for
SiHa cells than for the other cell
lines.4
Taken together, these results demonstrate that AdNIS is functional in
various tumor cell lines, whatever their origin.
AdNIS-infected Human Tumor Cells Are Efficiently Killed by
131I.
In vitro 131I uptake experiments were performed
on AdNIS-infected MCF7 and HT-29 cells to demonstrate that it was
possible to obtain cell killing with the AdNIS-radioactive iodide
system. Noninfected cells and cells infected with an adenoviral vector
encoding no transgene (AdCO1) were treated similarly, as controls.
After 131I treatment, clonogenic assays were performed, and
results are shown in Fig. 4
and expressed as the percentage of surviving cells. In each case, for
cells treated with bHBSS only, the numbers of colonies were comparable,
indicating that infection by an adenovirus (AdNIS or AdCO1) did not
affect cell survival. On exposure to 131I, around 30% of
the cells were nonspecifically killed, as assessed by the results
obtained for noninfected and AdCO1-infected cells. However, the number
of colonies recovered from AdNIS-infected cells was significantly lower
than that from noninfected or AdCO1-infected cells, showing a selective
killing effect of 131I on NIS-expressing cells. These
results thus demonstrate that coupling AdNIS and 131I
treatments in vitro efficiently and specifically leads to
cell killing, which is the end goal of the system.
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. As expected, a low amount of 125I was trapped by the
thyroid gland. In untreated control SiHa tumors, there was no
difference between the activity detected within the tumor or that
detected in its vicinity. On the contrary, for mice harboring an
AdNIS-treated tumor, the radioactivity detected within the tumor was
significantly higher than that in the control region, demonstrating
that AdNIS-treated tumors did specifically concentrate
125I. Similar results were obtained with MCF7 tumors,
confirming that the AdNIS vector is functional in vivo (data
not shown).
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. In each mouse, the
AdNIS-treated tumor accumulated significantly more radioactive iodide
than the control tumor. Indeed, the amount of 125I present
per milligram of tissue in the infected tumor was 4–25-fold higher
than that observed for the control tumor (Fig. 5B
; Table 1
). On average, the radioactive uptake (percentage of the total amount of
injected 125I) in the AdNIS-treated tumors was 11%/g tumor
tissue (range, 4.3–16.8%/g tumor tissue; see Table 1
for details). To
evaluate whether these radioactive iodide concentrations were
sufficient to affect tumor viability and/or growth, we repeated the
iodide uptake experiments as described above, but with
131I. Doses of 30, 60, or 90 µCi of 131I were
administered by i.p. injection to mice harboring an AdNIS-injected
tumor, and tumor volumes were followed for 14 days. Unfortunately, no
difference in tumor size could be observed between groups of animals
treated with AdNIS-131I and control groups treated with a
control virus or with AdNIS alone (data not shown). In addition,
histological analysis of all the tumors did not reveal any difference
in terms of necrosis. This absence of therapeutic effect of
131I indicates that despite an efficient iodide uptake (see
above), the radiation doses delivered in vivo to the tumors
were not high enough to affect tumor cell survival. Similar
observations have been made by Shimura et al.
(33)
working with transformed rat thyroid cells.
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. As expected, tissues naturally expressing NIS (stomach and thyroid) or
involved in iodide elimination (bladder) were visualized. The
AdNIS-infected tumor was clearly visible, with an intensity comparable
to that of the thyroid or the bladder. On the contrary, a noninjected
control tumor was not seen (data not shown).
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. Interestingly, these foci were generally located at the border between
necrotic and viable regions of the tumor (see Fig. 7, A–D
).
A possible explanation for this observation is that the adenoviral
preparation injected could diffuse more easily at the contact of softer
tissue (necrosis) and less so in viable areas. An alternative
hypothesis is that necrosis could be linked to a direct cytotoxic
effect of AdNIS infection. In some immunostained areas, it was clear
that labeling was confined to the cell membranes, demonstrating that
in vivo also, the AdNIS vector leads to correct localization
of the NIS protein (Fig. 7, E and F)
.
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DISCUSSION |
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In this study, we investigated the possibility of using an adenoviral vector to deliver the NIS gene into tumors, with the aim of treating them with radioiodine. We constructed a recombinant adenovirus, AdNIS, driving expression of the rat NIS gene under the control of the cytomegalovirus promoter. In vitro infection of human tumor cells from various origins, including mammary gland, prostate, lung, cervix, and colon, with AdNIS led to expression of a functional NIS protein, as revealed by efficient iodide uptake after infection. Iodide accumulation in AdNIS-infected cells was rapid and perchlorate sensitive, two characteristics previously reported for NIS-mediated iodide transport (9) . Depending on the cell line tested, iodide accumulation was 35–225-fold higher than that in noninfected control cells. In addition, AdNIS-infected cells were efficiently killed by 131I, as revealed by clonogenic assays. The AdNIS vector was also functional in vivo, as revealed by the iodide accumulation observed in vivo in tumors injected with AdNIS. Taken together, our results demonstrate that using the AdNIS vector is a valid approach to achieve efficient iodide uptake in tumors of nonthyroid origin.
While this study was in progress, transfer of the NIS gene with a retroviral vector was described in human [A375 (melanoma) and IGROV (ovarian carcinoma)] and mouse [CT26 (colon carcinoma) BNL.1 ME (transformed liver)] tumor cells (23) . In the cell lines tested, iodide uptake reached to a maximum 21-fold that observed with nontransduced cells and 35-fold that of cells transduced with a control retroviral vector (23) . Delivery of the NIS gene by an adenoviral vector is likely to be more efficient because the iodide uptake capacity of AdNIS-infected cells was increased up to 225-fold as compared with that of noninfected cells. Another major difference between both studies concerns the results obtained in vivo. Whereas Mandell et al. (23) induced tumors by injection of tumor cells first transduced in vitro with the NIS gene, we preferred to inject the AdNIS virus in established tumors, an approach required in a therapeutic situation. In that respect, adenoviral vectors present a clear advantage over retroviral vectors, namely, the ability to obtain concentrated preparations, which facilitates efficient in vivo gene delivery. Our results thus demonstrate for the first time that it is possible to transfer the NIS gene in a preformed tumor and to thereby confer a significant and relevant iodide uptake capacity in vivo.
The therapeutic efficacy of radioiodine is dependent on the radiation dose delivered to the target tissue (34) . The delivered radiation dose is proportional to both the effective half-life of 131I in the tumor [combination of the physical (8.02 days) and biological half-lives of 131I] and the radioactive concentration in the tumor, which is the ratio between the total uptake of iodide and the tumor mass. The maximal amount of 131I that can be given to a patient is limited by the radiation dose delivered to healthy tissues. To efficiently treat a tumor with 131I after transfer of the NIS gene, it is therefore important to combine in the tumor a high uptake of iodide, which depends on the level of NIS expression, with a long retention time of iodide, which is linked to the organification of iodide into cell proteins.
Intratumoral injection of AdNIS leads to efficient iodide uptake, since
uptake in treated tumors was up to 25-fold higher than that in control
tumors (Fig. 5
; Table 1
). Moreover, an average radioactive
concentration of 11% of the injected amount of 125I per
gram of tumor tissue was observed. This result is to be compared with
iodide concentrations of about 1% per gram of tissue in normal human
thyroid tissue and only 0.1% per gram of tissue or even less that are
routinely observed in human thyroid cancer tissues (35)
.
Importantly, such results were obtained even if NIS expression in the
tumor was not uniformely distributed, with foci of transduced cells
representing up to 30% of the tumor, whereas other areas were not
transduced (see Fig. 7
). This implies that the AdNIS vector could lead
to even higher iodide accumulation levels if the proportion of cells
expressing NIS after infection was increased. An interesting approach
in that respect is the modification of the viral capsid to improve the
capacity of the virus to infect the target cells (36, 37, 38, 39)
.
Higher transduction levels could also be achieved by injecting larger
doses of the virus at various points in the tumor. This would also
contribute to the homogeneity of NIS expression in the tumor, which is
quite important to consider for a therapeutic effect. Indeed, although
the ß emission of 131I can cover a short distance
(maximum, 2–3 mm) in biological tissues and thus deposit energy into
cells neighboring those expressing NIS, the radiation dose delivered
decreases rapidly with the distance (35)
, and the
bystander effect thus remains limited.
Another key issue is the retention of iodide in the target tissue
(3)
. Indeed, although the AdNIS-131I system
led to efficient cell killing in vitro (Fig. 4)
and despite
the high iodide concentrations reached in AdNIS-injected tumors
in vivo (Fig. 5B
; Table 1
), 131I administration
did not have any effect on tumor viability and growth in our study.
This observation is most probably linked to the fact that the tumors
did not retain iodide for a time period long enough to allow delivery
of a radiation dose affecting cell viability. In vitro data
confirmed that the tumor cells used in this study do not have the
capacity to organify and thus retain the iodide taken up after AdNIS
treatment (data not shown). This was similarly illustrated in a study
of tumors consisting of malignantly transformed rat thyroid cells that
had lost their capacity to concentrate iodide and that were transfected
in vitro with the rat NIS gene (33)
.
Although the tumors efficiently concentrated iodide, no effect of
131I on tumor growth was observed because the rapid iodide
efflux from the tumor did not allow the delivery of a radiation dose
sufficient to inhibit cell growth (33)
. Several advances
are conceivable to circumvent this lack of iodide retention in the
tumors. First, the efficiency of NIS gene transfer–and thus
the iodide uptake capacity of the target tissue–may be improved by the
use of modified vectors and/or higher viral doses (see above). Second,
the biological half-life of radioiodine in the tumor tissues could be
increased by coupling transfer of the NIS gene with delivery
of a gene involved in the iodide organification process, such as the
thyroperoxidase (1)
.
In view of the obtained results, coupling transfer of the NIS gene by an adenoviral vector and radioiodine administration appears to be a very promising strategy for treating tumors of various origins. Although improvements to achieve higher radiation doses in the target tissue will be required, application of this "targeted radiotherapy" approach in patients will be facilitated by our long-standing experience with radioiodine for thyroid cancer therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 A. B. is financed by a Marie Curie Research
and Training Grant from the Biotechnology research and technological
development program from the European Community. This work was
supported by grants from the Centre National de la Recherche
Scientifique, the Institut Gustave Roussy, Electricité de France,
and the Ligue Nationale Contre le Cancer. We acknowledge the support of
Associazione Italiana per la Ricerca sul Cancro (to S. F.). 
2 To whom requests for reprints should be
addressed, at Laboratoire de Vectorologie et Transfert de Gènes
UMR1582, Institut Gustave Roussy, 39 rue Camille Desmoulins PR2, 94805
Villejuif, France. Phone: (33)-1-42-11-50-82; Fax: (33)-1-42-11-52-45;
E-mail: boland{at}igr.fr
3 The abbreviations used are: NIS, sodium iodide
symporter; rNIS, rat NIS; FBS, fetal bovine serum; PFU, plaque-forming
unit(s); MOI, multiplicity of infection; ATCC, American Type Culture
Collection; bHBSS, buffered HBSS.
4 A. Boland, unpublished observations.
Received 12/13/99. Accepted 4/27/00.
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),
AdNIS-infected SiHa cells (•), and FRTL-5 cells (
). SiHa cells
were infected with AdNIS at a MOI of 10 for 29 h before the iodide
uptake experiment. Iodide uptake was measured by incubation for the
indicated time with 0.1 µCi of 125I. Results are
expressed in numbers of cpm for 7 x 105
cells and are representative of three separate experiments, each done
in duplicate.
