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Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Leslie J. DeGroot, M.D., Thyroid Study Unit, Mail Code 3090, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.
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Abstract |
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When AdCMVmIL2-infected mMTC cells were injected sc into their host animals, tumors developed in 2 of 10 animals, in contrast to 9 of 10 animals injected with AdCMVß-gal-infected mMTC cells and all 10 animals injected with parental mMTC cells. Moreover protected animals developed a long lasting immunity against mMTC tumor cells and their splenocytes, showing cytotoxicity to parental tumor cells, and active natural killer (NK) cell activity. BALB/c-SCID (severe combined immune deficiency) mice were also used to evaluate the function of NK cells in antitumor activities. No tumor developed in SCID mice injected with AdCMVmIL2-infected cells, whereas all animals injected with either AdCMVß-gal-infected or parental mMTC cells developed tumors.
Our data indicate that IL-2 production by MTC cells leads to rejection in syngeneic animals and suggest that both cytotoxic T cells and NK cells may play an important role. In addition, transduction of adenoviral vectors into tumor cells produces some nonspecific antitumor effects.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retroviruses have been used to transfer cytokine genes into tumor cells (10, 15, 16, 17) and induce protective antitumoral immunity after injection in syngeneic animals. A major concern with retroviral vectors is their genetic instability (18, 19, 20). It has been estimated that the mutation frequency can be as high as 0.5%/cycle of replication. Furthermore, as they integrate into cellular chromosomes, they could deregulate cellular functions or disrupt gene-coding regions. Another problem is that cell proliferation is necessary for gene transduction. High transduction efficiency and expression are difficult to obtain because the majority of cells in solid tumors proliferate poorly in vitro.
Adenoviral vectors are able to transduce genes into proliferating and nonproliferating cells and have been used for in vivo immuno- and gene therapy in many malignant diseases (21, 22, 23, 24). Adenoviral vectors have the advantage of having a broad host range, and potential capacity for large foreign DNA inserts. Furthermore, the safety of adenoviral vectors is well documented (25). Beginning in 1969, the U.S. armed forces administered an oral vaccine consisting of live unattenuated adenovirus 4 and adenovirus 7 in an attempt to prevent adenovirus 4- and 7-induced respiratory disease. Millions of people were immunized and protected against these illnesses. To date, no significant side-effects or illnesses have been related to the use of these vaccines (25). The adenovius genome rarely integrates into its host DNA, but, rather, persists extrachromosomally. This minimizes the risks of insertional oncogenesis and cellular gene activation (26). It is clear that adenoviral vectors have advantages in gene therapy of tumors.
Medullary thyroid carcinoma (MTC) is a tumor of the parafollicular (C) cells. It occurs in both sporadic and familial forms. Thirty to 50% of patients have lymph node metastases present at diagnosis. Treatment of metastatic disease with radiation is of uncertain benefit. The role of chemotherapy in MTC is also limited. Surgery is the only effective choice for treatment of tumor (27). However, many patients are left without tumor that can be identified by any imaging procedure after surgery, but with elevated calcitonin values, indicating the presence of tumor, and often ultimately develop fatally progressive disease. No effective salvage therapy exists for patients who fail the standard treatment. Therefore, new treatments are needed.
In this study, replication-defective adenoviral vectors transducing the Escherichia coli ß-galactosidase (lacZ; ß-gal) or murine interleukin-2 (mIL-2) gene were constructed. The effectiveness of adenoviral vectors for ex vivo gene transduction into MTC cells was examined. We also tested the AdCMVmIL2-directed expression of functional mIL-2 in infected MTC cells and evaluated tumor vaccination therapy using transduced mMTC cells in a mouse model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of recombination-defective adenoviral vectors
Replication-defective adenoviral vectors containing mIL-2 or
lacZ gene under transcriptional control of the human
cytomegalocyte viral immediate early promoter/enhancer system were
constructed according to the protocol previously reported (25).
Briefly, the mIL-2 gene was subcloned from pZIPNEOSV/mIL2 into the
eukaryotic expression vector pCA13 at the BamHI site. pCA13
plasmid contains Ad5 sequences from 22 bp (0 map units) to 5790 bp
(16.1 µ) with a deletion of E1 sequences from 342-3523 bp (1.0–9.8
map units). A multiple restriction site was present at the position of
the deletion for cloning inserts under the control of the human
cytomegalocyte viral immediate early promoter (-299 to +72), and the
simain virus 40 polyadenylation signal was inserted into the E1 region
(30). The expression cassette pCA/mIL2 and the plasmid pJM17 were
cotransfected into 293 cells by the calcium phosphate precipitation
method. Incorporation of the expression cassette into the isolated
recombinant virus was confirmed by codigestion with restriction enzymes
BamHI and an enzyme (HindIII) unique in mIL-2
cDNA. This helped to determine the correct direction of insertion of
mIL-2 cDNA. The correct virus construct was also confirmed by the
expression of functional mIL-2 in infected cells using an
IL-2-dependent CTLL-2 proliferation assay (described below).
The adenoviral vector-expressing lacZ gene was constructed using the protocol described above, except that the ß-gal gene was inserted into EcoRI and BamHI sites. Viruses were screened by analysis of the enzyme digestion map of viral DNA with enzyme EcoRI and BamHI. Correct virus was confirmed by 4-Cl-5-bromo-3-indolyl-ß-galactosidase (X-Gal) staining (described below). This virus was used to evaluate the efficiency of gene transfer into the MTC cells and as a control in animal studies.
Rescued virus was plaque purified and amplified in 293 cells. The viral preparations were purified by two CsCl density centrifugations, dialyzed, and stored in 20% glycerol at -70 C. The titers of the viral stocks were determined by plaque assay using 293 cells (31, 32).
Assay of mIL-2 using CTLL-2 cells
Murine IL-2 bioactivity was measured by a standard bioassay
protocol using an IL-2-dependent murine T cell line, CTLL-2, as
described previously (33). The transduced cells were cultured at 1
x 106/ml for 24 h, and supernatant was saved for
mIL-2 assay. CTLL-2 cells (5 x 103) were incubated
with a mIL-2-containing sample in a volume of 180 µl for 24 h at
37 C in 96-well microtiter plates. Then [3H]thymidine
(0.5 µCi/well) was added, and the incorporation of radiolabeled
thymidine into DNA was determined after overnight incubation.
ß-Gal histochemistry
ß-Gal activity was assayed in tumor cells after infection with
AdCMVß-gal, using histochemical methods (34, 35). Infected
cultured cells were washed with PBS, pH 7.2, and then fixed in 4%
paraformaldehyde in PBS for 15 min. The cells were washed with PBS
three times and overlaid with a histochemical reaction mixture
containing 1 mg/ml X-Gal, 5 mM potassium ferricyanide, 5
mM potassium ferrocyanide, and 2 mM
MgCl2 in PBS. The X-Gal was dissolved in dimethoxysulfoxide
at 40 mg/ml and then diluted into the reaction mixture just before use.
Reaction was continued for 14–18 h by incubating the reaction system
at room temperature. ß-Gal-positive cells showing a blue nuclear
color were counted, and the percentage of positive cells was
calculated.
Infection of MTC cells with AdCMVß-gal or AdCMVmIL2
MTC cells were harvested during exponential growth of the cell
culture. The cells were washed with serum-free medium, counted, and
centrifuged (1000 rpm for 5 min). Viability was examined by trypan blue
dye exclusion and always showed more than 95% living cells.
AdCMVß-gal or AdCMVmIL2 was added to the cell pellets at a
multiplicity of infection (moi) of 500 plaque-forming units (pfu)/cell
and incubated for 2 h at 37 C in a minimum volume (usually 500
µl) of infection medium (containing 2% FBS) to permit efficient
infection (standard infection condition). Cells were then washed with
fresh complete medium three times to remove free viral particles,
resuspended in fresh complete medium, and incubated at 37 C. The
supernatants were removed for the detection of mIL-2 activity in the
CTLL-2 proliferation assay after 24-h incubation, and cells were fixed
and stained for ß-gal activity to estimate the efficiency of gene
transfer after 48-h incubation. When the duration of expression of
mIL-2 was examined in transduced mMTC cells in culture, RPMI 1640
medium containing 5% FBS was used.
Direct cytotoxicity of vectors to infected cells was examined. After 72-h incubation of infected cells, a single cell suspension was prepared, and viable cells were counted using trypan blue exclusion.
To optimize transduction conditions, individual experiments varied the volume of the medium used for transduction, the moi (0–1000), or the period of exposure to virus.
Tumor production
Mouse MTC cells were washed in RPMI medium, counted, and
injected sc into syngeneic mice at a dose of 2 x
106/mouse. The injected mice developed palpable tumors
after 12–14 days, and tumors continued to grow. When infected mMTC
cells were injected, the animal was considered tumor free when no
palpable tumor was detected during the experimental period (at least 2
months). When BALB/c-SCID (severe combined immune deficiency) mice were
used, a dose of 1 x 106 cells was used to develop
tumors.
Distant site challenge with parental mMTC cells in tumor-free
mice
Tumor-free mice injected with AdCMVmIL2-infected mMTC cells were
challenged with wild-type mMTC cells at a different site after 60 days.
The same cell number was used as that in the first injection.
Calculation of the volume of sc tumors
Subcutaneous tumor volumes were determined from the formula
v = a2b/2, where a is
the shortest diameter, and b is the longest diameter of the
tumor. Tumor growth was measured every 2 or 3 days.
Cell-mediated cytotoxicity assays
The nonradioactive cytotoxicity assay kit (Promega,
Madison, WI) was used to evaluate cell-mediated cytotoxicity. This
assay quantitatively measures lactate dehydrogenase (LDH), a stable
cytosolic enzyme that is released upon cell lysis, in much the same way
as the standard chromium release assay. Released LDH in culture
supernatants is measured with a 20-min coupled enzymatic assay that
results in the conversion of a tetrazolium salt into a red formazan
product. The amount of color formed is proportional to the number of
lysed cells.
To measure tumor-specific CTL, mice were immunized twice with AdCMVmIL2-transduced mMTC cells. Two weeks postimmunization, splenocytes were collected and stimulated in vitro by incubating 3 x 106 effector cells/ml with 2 x 105 mitomycin-C-treated tumor cells/ml in 75-ml flasks for 5 days at 37 C in the presence of 20 U/ml recombinant IL-2. As a control, splenocytes from nonimmunized mice were also collected and used in the assays. Target cells (1 x 104) were mixed with stimulated effector cells at final effector/target (E:T) ratios between 100:1 and 6.25:1 in 96-well U-bottom plates. The plates were lightly centrifuged at 500 rpm for 4 min and incubated for 4 h at 37 C in 5% CO2. The plates were then centrifuged at 500 rpm (250 x g) for 4 min, and 50-µl aliquots of supernatants were transferred from all wells to a fresh 96-well flat-bottom plate. Fifty microliters of substrate were added to each well and incubated for 20 min at room temperature. Fifty microliters of stop solution were added, and the absorbance was recorded at 490 nm.
The percentage of specific lysis was calculated using the formula: % cytotoxicity = (experimental LDH release - effector cell spontaneous LDH release - target cell spontaneous LDH release)/(target cell maximum LDH release - target cell spontaneous LDH release).
NK assays were performed using the NK-sensitive cell line YAC-1 as the target cell.
Animals
Inbred 4-week-old female BALB/c and BALB/c-SCID mice were
purchased from Jackson Laboratory (Bar Harbor, ME) and maintained at
the Carlson Biocontainment Suite (The University of Chicago, Chicago,
IL) under standard conditions, according to the guidelines of the
Animal Research Center.
Statistical calculations
Student’s t test was used to analyze the data.
P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, all three MTC cell lines show a
moi-dependent transduction efficiency. With a decrease of moi from 800
to 200, the positive staining of infected cells decreased from 62.7%
to 29.0% for the human MTC cell line, from 37.7% to 9.3% for the rat
MTC (rMTC) cell line, and from 78.0% to 30.7% for the mMTC cell line.
No ß-gal activity was found in uninfected cells, indicating that
endogenous ß-gal activity was not present in these cells under our
reaction condition.
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). AdCMVß-gal
vector was also examined, and a similar result was obtained (Fig. 1B).
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), probably due to the loss of virus as
cells replicate and some die. We believe that the level of secreted
mIL-2 is enough to affect the immune system when the infected cells are
injected into syngeneic animals.
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). No X-Gal staining was found in
unexposed cells, whereas significant staining over baseline was seen at
an moi of 25 for both rMTC and mMTC cells. The positively staining
cells increased with infection moi. A moi of 500 was chosen for
in vitro infection studies.
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). Virus concentration was also evaluated for
efficiency of transduction. rMTC and mMTC cells were exposed to
AdCMVß-gal for 2 h using 500 moi, suspended in various amounts
of transduction medium (100–1000 µl). As shown in Fig. 3C, as the
volume of infection solution increased from 100 to 1000 µl;
positively staining cells decreased from 70.5% to 16.75% for the rMTC
cell line and from 73.75% to 23.5% for the mMTC cell line.
Expression of mIL-2 after intratumoral administration of AdCMVmIL2
in mice
To verify the expression of mIL-2 in infected tumor cells in
vivo, treated tumors (3–4 mm) injected with 1 x
109 pfu in 100 µl with either AdCMVmIL2 or AdCMVß-gal
were excised 3 days after the injection. A single cell suspension was
prepared, primary cultures were made at 2 x 106
cells/ml, and cells were cultured overnight in complete RPMI 1640
medium. Supernatants were then saved, and equal aliquots of the
supernatants were taken to determine the presence of mIL-2 using the
IL-2-dependent CTLL-2 proliferation assay. No proliferation of CTLL-2
cells was observed with supernatants from uninfected tumors or tumors
treated with AdCMVß-gal, whereas supernatants from AdCMVmIL2-treated
tumors induced a high level of proliferation (Fig. 4), indicating the production of
functional mIL-2 by the AdCMVmIL2-treated tumor cells. It should
be noted that in vivo, the expression of mIL-2 by infected
tumor cells might stimulate immune cells to produce mIL-2. The in
vitro measurement of the production of mIL-2 by in vivo
infected tumor cells largely excludes the involvement of the host
immune system. The mIL-2 production in vitro thus does not
necessarily reflect the in vivo situation.
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).
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, no mouse developed a tumor,
showing that a long lasting antitumor immune response was established
against subsequent challenge.
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).
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. Antitumor lytic activity in BALB/c
mice immunized with AdCMVmIL2-transduced mMTC cells was far superior to
that in control animals (65.4% vs. 4.4% lytic activity at
an E:T ratio of 100:1 compared to 28.1% vs. 0 at an E:T of
25:1). No lytic activity was detected at an E:T ratio lower than 25:1
in control animals, whereas more than 17% specific lytic activity was
detected at an E:T ratio of 6.25:1 in immunized animals. Lytic activity
against YAC-1 target cells was also superior in immunized animals
compared with that in control animals. This is probably because NK
cells were already activated by mIL-2 in immunized animals, and after
in vitro stimulation with rIL-2 they had increased ability
to lyse NK-sensitive YAC-1 cells. These results indicate that both
specific (T cell) and nonspecific (NK cell) antitumor activities exist
in AdCMVmIL2-treated animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our study confirms and extends these findings. Our results demonstrate that replication-defective adenoviral vector can transfer foreign genes into MTC cell lines both in vitro and in vivo. The efficiency of transduction in vitro is dependent upon moi, duration of exposure to the vector, and concentration of the vector. Optimizing these conditions resulted in a good transduction efficiency and expression of the lacZ gene in MTC cells. It should be noted that transduction efficiency is probably underestimated by using X-Gal staining. Under conditions optimized for in vitro histochemistry, up to 20% of cells stably transfected with lacZ gene remained unstained by X-Gal compared with immunostaining using anti-ß-gal antibodies. During in vivo studies, 3 times more positive cells were detected by the anti-ß-gal antibody than by X-Gal staining after transduction with an adenoviral vector (41).
Local secretion of mIL-2 by transduced tumor cells not only abrogated
the tumorigenicity of these cells in immunocompetent (BALB/c) and
immunodeficient (BALB/c-SCID) mice, but also induced a long lasting
state of antitumoral immunity in immunocompetent mice, suggesting that
both specific and nonspecific immunities play a role in antitumor
activities. We found that the tumor sizes were smaller in
AdCMVß-gal-infected groups than in uninfected groups in both BALB/c
and SCID mice (data not shown), and one mouse in the AdCMVß-gal
infected group of BALB/c mice did not develop tumor, suggesting that a
nonspecific viral effect exists. This is not a direct cytotoxic effect,
because our vectors have no direct cytotoxic effect on infected cells
(Fig. 1).
We hypothesize that the nonspecific antitumor effect comes from the virus. Infected tumor cells that express antigens from the viral vectors will activate host immunity and be easily attacked by host immune effector cells, including NK cells. NK cells do not require prior contact with target antigens to develop cytolytic capacities, as they naturally possess the ability to kill certain cells infected by virus. Killing by NK cells is not specific and not restricted by MHC molecules (42). This nonactivated NK activity has only a limited effect and could not stop the development of tumor. After activation by mIL-2 secreted by transduced tumor cells, NK cells have an increased ability to kill the injected tumors. This might be the main mechanism through which SCID mice reject tumors.
We also demonstrate that adenovirus vectors are capable of directly transferring the IL-2 gene to the tumor cells in animals, thus avoiding ex vivo manipulations of the cells. It may be more practical to treat an established tumor in vivo than to use ex vivo manipulation. This method led to a complete regression of tumor in 50–75% of treated mice with an initial tumor 2–5 mm in diameter, and the successfully treated animals developed a long lasting state of antitumor challenge. For large tumors (8–12 mm in diameter), stabilization of tumor size was obtained (23). However, there was a vast difference in the effect of IL-2 gene-based immunotherapy depending on the model under study (43). Some reports showed only a local therapeutic effect on preexisting tumors and may provide some benefit for preventing tumor recurrence or micrometastasis (24, 43). We are now performing an in vivo study using our viral vector, examining the antitumor effect in preestablished tumors in an animal model.
Combination therapy has been reported for some cancers (42, 43, 44, 45, 46, 47). A significant antitumor effect on preexisting tumors was obtained after treatment with combinations, including IL-2, IL-12, and/or the herpes simplex virus thymidine kinase gene.
We have already constructed other replication-defective adenoviral vectors harboring IL-12, thymidine kinase, and B7.1, and we will test different combinations on preexisting sc tumors and evaluate their therapeutic potentials.
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Acknowledgments |
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Received August 26, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IFN-
, and TNF-. Lab Invest 74:146–157[Medline]
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