M. Reincke 1 D. Ortmann2 J. Hausmann 3 F. Beuschlein2
Abstract
Adrenocortical carcinoma (ACC) is still one of the most devastat- ing human tumors with a five year survival as low as 20%. In a previous study, we showed that DNA vaccination followed by vaccinia virus was able to break immune tolerance against mu- rine steroidogenic acute regulatory (mStAR). Prophylactic vacci- nation in syngenic mice resulted in protective immunity against Sp2-0 tumor cells expressing mStAR. However, approximately a third of the animals developed tumors despite vaccination. This prompted us to investigate whether vaccination failure is responsible for this phenomenon. BALB/cBALB/c mice (in groups of 6-9 animals) were vaccinated intramuscularly by injection of cDNA expression vectors encoding mStAR three times at weekly intervals. This was followed by a recombinant vaccinia virus (rVV-mStAR) infection to boost immune response. Ten days after the last vaccination, Sp2-mStAR or parental Sp2-0 cells (as con-
trols) were injected s.c. Tumor development was monitored by daily palpation. Approximately two weeks later, the animals were sacrificed and the spleens removed. After restimulation with the cell lines expressing mStAR, the splenocytes were tested for presence of mStAR self-reactive cytotoxic T-lymphocytes using ELISPOT analysis. With this approach, we were able to show that those animals protected from tumor growth had a specific T-cell response against StAR whereas mice without a specific T-cell response developed Sp2-mStAR tumors. Our data demonstrate that vaccination failure, probably due to the low an- tigenicity of mStAR, is responsible for tumor growth in our model system.
Key words
Adrenocortical carcinoma · Steroidogenic acute regulatory pro- tein . Immunotherapy . Mouse . CTL . Tolerance
Introduction
Adrenocortical carcinomas (ACCs) are highly malignant but rare endocrine neoplasms [1]. Analysis of clonality using X-chromo- some inactivation analysis demonstrated that ACCs are monoclo- nal in origin [2]. Comparative genomic hybridization identified an unusual high rate of chromosomal losses and gains that cor- related well with the tumor’s aggressive biological behavior [3]. The most frequent genetic aberration in ACC is IGF-II overexpres-
sion associated with mutations in chromosome 11p15.5, but also mutations within the p53 gene locus and other oncogenes and tumor suppressor genes have been reported [4]. While the ma- jority of ACCs are sporadic, recent studies from Brazil point to germline cell mutations within the p53 gene as a cause of ACC in certain high risk populations. The exclusive adrenal pheno- type of these p53 mutations is explained by pH-dependent acti- vation of the mutation within the adrenal cortex [5].
Affiliation
1 Medizinische Klinik - Innenstadt, Klinikum der Maximilians-Ludwigs-Universität München
2 Department of Internal Medicine II, Division of Endocrinology, University Hospital of Freiburg 3 Institute for Medical Microbiology and Hygiene, Department of Virology, University of Freiburg, Freiburg, Germany
Correspondence
Martin Reincke, M. D. · Medizinische Klinik - Innenstadt · Klinikum der LMU München · Ziemssenstr. 1 .
80336 München · Germany · Phone: +49 (89) 51 60 21 00 · Fax: +49 (89) 51 60 44 28 .
E-Mail: martin.reincke@med.uni-muenchen.de
Received 8 January 2004 . Accepted after revision 10 March 2004
Bibliography
Horm Metab Res 2004; 36: 411-414 @ Georg Thieme Verlag KG Stuttgart . New York .
DOI 10.1055/s-2004-814571 . ISSN 0018-5043
Most adrenocortical carcinomas are larger than 5 cm at time of diagnosis, and mean tumor weight is around 800 grams [5]. Sev- enty percent of ACC patients suffer from an advanced tumor stage with local invasion or distant metastasis at diagnosis [6], resulting in low treatment success rates even when complete surgical resection is possible. Response rates to cytotoxic sys- temic chemotherapy and mitotane adrenolytic agent range from 10 to 50% [7], but time to relapse is short, and the overall 5 year survival rate averages around 20%. Improved treatment strate- gies are clearly required for this carcinoma, and include immu- notherapeutic strategies such as vaccination against tumor- specific antigens.
So far, no tumor specific antigen as a potential target for immune therapy in ACC has been identified. However, adrenocortical car- cinomas express certain steroidogenesis-related receptors and enzymes that are relatively specific to the adrenal and might be used as targets. In a recent study, we identified the steroidogenic acute regulatory protein (StAR) as a promising antigen [8]. Based on this evidence, we evoked a specific immune response against murine StAR protein using a DNA-based immunization approach in a syngenic mouse adrenocortical carcinoma model [9]. Immu- nity to StAR was successfully induced in the majority of mice. However, approximately 30% developed StAR-expressing marker tumors despite vaccination. The purpose of this study was to in- vestigate whether vaccination failure is responsible for missing protection against StAR-expressing tumor cells.
Materials and Methods
The tumor model and the techniques used in this study have re- cently been described in detail [9]. In brief, the mouse myeloma cell line Sp2-0 (ATCC CRL1581) was grown in DMEM supple- mented with 20% heat-inactivated fetal bovine serum. Sp2-0 cells were stably transfected using an expression construct en- coding mouse StAR [10] and mouse P450scc [11] and selected for stable expression of transgenes by addition of 100 mg/ml Zeocin into the culture medium. Female BALB/c mice were ob- tained from Charles River Laboratories (Sulzfeld, Germany). Sub- cutaneous injection of mice with Sp2-mStAR cells or (as a con- trol) parental Sp2-0 cells resulted in palpable tumors after 8 - 10 days with a penetrance of >90%. The protocols used were ap- proved by the institutional animal review committee.
Mice were immunized three times in weekly intervals by co-in- jecting plasmid constructs of pSecTagA-mStAR (eukaryotic se- cretion vector expressing mStAR), pRJB-GM (plasmid expressing IL-12p70), and pApIL-12p70 (plasmid expressing GM-CSF) into the tibialis anterior muscle. One hundred micrograms of pSec- TagA-mStAR and 25 µg each of plasmids pRJB-GM and pApIL- 12p70 were applied in a total volume of 100 ul H20. The recombi- nant vaccinia virus encoding mStAR (VV-mStAR) or mP450 side- chain cleavage enzyme (VV-mP450scc) was delivered i.v. at 5 × 106 p.f. u. per mouse 10 days after the last DNA immunization.
ELISPOT
MultiScreen HA sterile plates (MAHAS4510; Millipore, Eschborn, Deutschland) were coated with 1 µg of anti-mouse IFN-y capture antibody (clone R4-6A2; PharMingen) in 100 ul of PBS overnight
at 4℃. After blocking for 2 h with 200 ul of medium (DMEM, 5% FCS and penicillin and streptomycin), splenocytes were seeded in serial fourfold dilutions from 1000 000 to 125 000 cells per well in 100 ul of medium including a negative control and a well re- ceiving 7.5 µg/ml Concanavalin A plus cells (positive control). After 24 h at 37 ℃, the cells were removed and the biotinylated anti-mouse IFN-y antibody was added (clone XMG1.2; PharMin- gen) for 12 hours at 4℃. After washing, the wells were incubated with 100 ul of streptavidin-alkaline phosphatase (PharMingen) for 1 h at room temperature. After washing, 3-amino-9-ethylcar- bazole substrate (Sigma) was added. The reaction was interrupt- ed by rinsing the wells with water. Spots were quantified in an ELISPOT reader (BIOSYS; Karben; Deutschland).
Results
In the present study, we have analyzed the CTL response against mStAR in our tumor model. BALB/c mice were immunized 3 times in weekly periods with a plasmid encoding a secreted full length form of mStAR followed by a booster infection with re- combinant vaccinia virus-encoding mStAR (group A; experimen- tal design, see Fig. 1). After tumor seeding, we compared the im- mune response in those animals that developed tumors despite specific immunization (n = 9, 53%) with the immune response of those animals protected from tumor development by vaccination (n=8, 47%). A control group was vaccinated with empty vector pSegTag followed by infection with recombinant vaccinia virus encoding mP450scc (group B), all of which developed tumors (n = 15, 100%).
Two weeks after tumor cell seeding, splenocytes were analyzed for ex vivo mStAR-specific CTL frequencies using the IFN-y ELI- SPOT technique after 24 h of in vitro stimulation using syngenic Sp2-mStAR myeloma cells. Using this approach, a reproducible CTL response was detectable against mStAR in those animals of group A that did not develop any tumors (Fig. 2). At the same time, mice from group A that developed Sp2-mStAR tumors only had a weak CTL response similar to background (Fig. 3). These data agree with the notion that vaccination failure in group A animals is responsible for tumor growth.
Discussion
Tumors such as ACC are highly malignant with low response rates to classical therapies such as polychemotherapy and radia- tion therapy. Novel anti-tumor strategies include gene therapy using the suicide gene approach with adenoviral and retroviral vectors, immunotherapy with DNA vaccination and antigen-pre- senting dendritic cells (DCs) or a combination of these regimens. These strategies have been extensively tested in rodent models and have also been used with limited success in phase II and III trials on humans suffering from melanoma, lymphoma and pros- tate cancer [12-15].
Specific tumor antigens have not been yet identified in endocrine tumors. Two approaches for DC vaccination have recently been used - first, preparations of autologous tumor cells as antigen delivery within a DC based therapy scheme. In pilot studies on
4 weeks old BALB/c
3x DNA vaccination (DDD)
booster infection with recombinant Vaccinia Virus (rVV)
transplantation of tumor cells
CTL
Tumor: n=9
Group A n=17
pSec TagA-mStAR
rVVmStAR
o
Sp2-mStAR
No tumor: n=8
Group B n=15
pSecTagA
rVVmSCC
Sp2-mStAR
Tumor: n=15
0
1
2
3,5
5
7weeks
Figure 1, Reincke et al.
number of spot-forming cells /
25
Group A: DDDStAR + rVV-mStAR + Sp2-mStAR (n=8)
Group B: DDDpSecTagA + rVV-mP450scc + Sp2-mStAR (n=9)
10 splenocytes
20
15
10
5
I
I
0
Stimulator cells:
Sp2-mStAR
Sp2-0
Subcutaneous tumors: -
+
-
+
Figure 2, Reincke et al.
neuroendocrine pancreatic carcinomas and parathyroid carcino- mas in advanced disease stages, this resulted in tumor lysate- specific anti-tumor immunity [16,17]. Second, cell-specific anti- gens, such as specific proteins involved in the synthesis of hor- mones or hormones themselves can be used to induce a cytotox- ic T-cell response. This approach has already been reported as successful in inducing a Th1-dominated cellular immune and
number of spot-forming cells /
25
Group A: DDDStAR + rVVmStAR + Sp2-mStAR (n=9)
20
Group B: DDDpSecTagA + rVV-mP450scc+ Sp2-mStAR (n=6)
10 splenocytes
15
10
5
T
I
0
I
T
Stimulator cells:
Sp2-mStAR
Sp2-0
Subcutaneous tumor:
+
+
+
+
Figure 3, Reincke et al.
clinical responses in metastasized medullary thyroid carcinoma [18,19].
Based on these results, a multitude of adrenocortical enzymes such as 11ß-hydroxylase (P450c11), 21-hydroxylase (P450c21) and P450scc (side-chain cleavage enzyme) as well as regulatory proteins such as steroidogenic factor 1 (SF-1) and DAX-1 might serve as specific targets of a cytotoxic immune response in ACCs. Many of these antigens are expressed in adrenocortical tu- mors. StAR, which is responsible for the transport of cholesterol through the outer to the inner mitochondrial membrane during the process of steroid biosynthesis, presents an interesting target for anti-tumor immune response. We have demonstrated that
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StAR is not only expressed in adrenocortical adenomas but also in carcinomas [8], so it can be used in an immunotherapeutic ap- proach.
As proof of principle, we recently investigated StAR as a potential target for an immunization-based therapy in a syngenic mouse tumor model using DNA vaccination [9]. Since the immunogenic epitopes of the protein were not known, we subcloned the full- length cDNA into the DNA expression vector pSecTagA. BALB/c mice were vaccinated with cDNA expression vectors encoding for StAR or an empty vector (pSecTagA) three times in three weeks as controls followed by vaccinia virus vaccination in a prime-boost approach. This vaccination strategy induced a specific T-cell response as measured by ELISPOT and was protec- tive against syngenic Sp2-mStAR cell tumor growth.
In the present study, we extended our investigation further to study the phenomenon of tumor growth which occurred despite specific vaccination against mStAR. Theoretically, different mechanisms may be responsible for this phenomenon: First, the Sp2 cells could lose the antigen, mStAR. Second, the cytotoxic immune response induced by vaccination might be too weak to prevent Sp2-mStAR cell seeding, and third, there could be a com- plete failure of the vaccination scheme in some but not all ani- mals. In our previous report, we excluded loss of antigen expres- sion in most of the tumor tissues analyzed [9]. The data present- ed here indicate vaccination failure in those animals that devel- oped mStAR tumors. Overall, the immune response against the selected antigen mStAR appeared rather weak, although direct comparison against other antigens was not part of our protocol. Animals developing tumors had no measurable T-cell response against mStAR (Fig. 3). In contrast, animals protected from tumor development had a positive T-cell response (Fig. 2). Taken to- gether, these data indicate that we failed to evoke a specific T- cell response in some animals with the vaccination schedule used in this study. Whether low antigenicity of mStAR or techni- cal problems with application of the vaccine were responsible has to be elucidated.
In summary, we have shown here that immunological tolerance against mStAR can be broken using DNA vaccination followed by recombinant vaccinia virus infection. However, prophylactic im- munization is not effective in all animals, which is mainly due to vaccination failure. These results warrant future research on fur- ther enhancement of T-cell response to adrenal-specific antigens in ACCs.
Acknowledgements
This study was supported by a grant from the Dr. Mildred Scheel Stiftung to F.B and M.R., and by the Deutsche Forschungsgemein- schaft (Re 752/11-1)
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