Abrogation of TLR4 and CD14 Expression and Signaling in Human Adrenocortical Tumors
Waldemar Kanczkowski, Piotr Tymoszuk, Monika Ehrhart-Bornstein, Manfred P. Wirth, Kai Zacharowski, and Stefan R. Bornstein
Departments of Medicine III (W.K., P.T., M.E .- B., S.R.B.) and Urology (M.P.W.), Technical University of Dresden, 01307 Dresden, Germany; and Clinic of Anesthesiology, Intensive Care Medicine, and Pain Therapy (K.Z.), 60590 Frankfurt am Main, Germany
Context: Adrenocortical carcinoma (ACC) is a rare tumor with poor prognosis. The expression of innate immunity receptor Toll-like receptor 4 (TLR4) was recently reported in various human tu- mors, and TLR4 was shown to regulate tumor immune escape processes, proliferation, and resis- tance to chemotherapeutical agents.
Objective: The aim of this study was to investigate TLR4 expression, signaling, and function in the process of tumorigenesis in the human adrenal cortex.
Measurements and Main Results: Real-time PCR analysis of human ACC (n = 8), adenoma (n = 8), and ACC cell lines (SW13, NCI-H295R, and HAC15) revealed a significant down-regulation of TLR4, MD2 (myeloid differentiation protein-2), and cluster of differentiation 14 (CD14) mRNA compared with normal human adrenal cortex and adrenocortical cells in primary culture. Furthermore, im- munohistochemistry revealed an abrogation of TLR4 and CD14 expression in ACC but not adenoma tissues. Western blot analysis of MAPK, AKT, activator protein-1, and nuclear factor-KB signaling revealed that the ACC cell lines are unresponsive to lipopolysaccharide action. Restoration of TLR4 signaling by stable transfection of TLR4-CD14 plasmid into NCI-H295R cells sensitized them to lipopolysaccharide incubation as shown by nuclear factor-KB activation and decreased cell viability and induced apoptosis in these cells.
Conclusion: Our results demonstrate a significant reduction in the expression of TLR4 and CD14 and an inactivation of TLR4 signaling in ACCs. Furthermore, our data show that reintroduction of TLR4 expression in ACCs may provide a novel therapeutic strategy for adrenal cancer. (J Clin Endocrinol Metab 95: E421-E429, 2010)
B enign adrenal masses are common endocrine tumors with a prevalence of at least 3% in the population aged older than 50 yr (1). In contrast, adrenocortical car- cinoma (ACC) is a rare malignancy with an incidence of one to two per 1 million and a variable but generally poor prognosis (2). The etiology and molecular pathogenesis of ACC is poorly understood; however, inactivating muta- tions at the 17p13 locus including the TP53 tumor sup- pressor gene and alterations of the 11p15 locus leading to IGF II overexpression are common in patients with ACC (3, 4). Because clinically unapparent adrenal masses are
frequently found incidentally using modern diagnostic technologies such as computer tomography, distinguish- ing malignant from the benign adrenal tumors remains a clinical dilemma (5).
Few reliable markers allowing a clear-cut histological differentiation of benign and malignant adrenal lesions exist. Early and accurate diagnosis of ACC is, however, extremely important because adrenocortical carcinoma are resistant to chemotherapeutical treatment due to high expression of multidrug-resistance protein or P-glycopro- tein (6). Mitotane therapy, which is the only Food and
Abbreviations: ACC, Adrenocortical carcinoma; ADN, adenoma; CD14, cluster of differ- entiation 14; ITS, insulin-transferrin-sodium selenite; LPS, lipopolysaccharide; nAdr, normal adrenal gland; NF-KB, nuclear factor-KB; TLR, Toll-like receptor; TUNEL, terminal deoxy- nucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.
Drug Administration-approved drug for ACC treatment, only modestly prolongs life expectancy, which is only 5 yr in advanced ACC tumor stages (7); therefore, other ther- apeutic strategies must be considered. One promising and novel strategy is an immune therapy designed to break a state of immune tolerance to eliminate tumor cells. How- ever, the low number of ACC-specific antigens (especially in undifferentiated tumors) and insufficient knowledge of the mechanisms underlying this immunosuppressive en- vironment have made the design of such a study very dif- ficult (8) and may at least partially explain the failure of an initial study performed in two patients with ACC, which were vaccinated with autologous dendritic cells pulsed with autologous tumor lysate (9). Recently a National Institutes of Health international consensus conference on adrenal cancer held in Ann Arbor (Michigan) stressed the need to identify new reliable markers and therapeutic reg- iments for the clinical management of adrenal tumors (1). We have been focusing on the role of immune adrenal interactions in the regulation of adrenal hormone produc- tion and cell proliferation and have shown that adreno- cortical adenomas have abnormal expression of cytokine receptors, which influences the tumor cell proliferation. Moreover, we have shown an abrogation of constitutive major histocompatibility complex class II expression in adrenocortical carcinomas, which was associated with a malignant phenotype (10, 11).
Toll-like receptors (TLRs) including TLR4, the recep- tor for Gram-negative bacterial lipopolysaccharide (LPS), are type I transmembrane receptors widely expressed by cells of the innate and adaptive immune system. More recently, expression of several TLRs including TLR4 has also been found in nonimmune parenchymal tissues in- cluding cells of the endocrine system (12). We previously demonstrated an expression of several TLRs in normal human adrenal cortex and the primary adrenocortical cell cultures (13). Using TLR2- and TLR4-deficient animals, we demonstrated that the expression of these receptors is critical for a proper functioning of the immune-adrenal cross talk and hence adequate glucocorticoid secretion during systemic-inflammatory response syndrome-like conditions (14, 15). Interestingly, evidence increasingly correlates TLR4 expression and signaling with cell pro- liferation. We have shown that TLR4 deficiency promotes hypertrophy of murine adrenal cortex (15), whereas in other studies TLR4 signaling was found to restrict the proliferation of retina progenitor cells (16). Numerous studies have reported the expression of functional TLRs in malignantly transformed cells, providing a novel feature in the study of immune-tumor interactions, especially the mechanism responsible for the immunosuppressive envi- ronment that characterizes many tumors. The relationship
between inflammation and tumorigenesis is widely ac- cepted, and the expression and activity of TLR4 may play an important role in this process. Indeed, an overexpres- sion and hyperactivation of TLR4 has recently been re- ported in ovarian, prostate, lung, head, and neck squa- mous tumors. In these tumors activation of TLR4 promoted tumor evasion from immune survivalence, en- hanced cell proliferation and chemokine production, and induced the secretion of immunosuppressive cytokines and resistance to TNF-a and TRAIL (TNF-related apop- tosis-inducing ligand)-induced apoptosis (17-19).
Therefore, in the present study, we aimed to elucidate the potential role of TLR4 in the process of adrenal cortex tumorigenesis by analysis of its expression and signaling in adrenocortical carcinoma cell lines and tissues. Under- standing the function of TLR4 in human adrenocortical cancer cells may help to improve potential therapeutic strategies including immunotherapy.
Materials and Methods
Patient samples
Specimens were obtained from 16 patients (10 females and six males; mean age 52.19 yr). Surgically removed adrenocortical incidentaloma tissues were further analyzed according to the criteria of Weiss (20). Adrenocortical tumors removed from eight patients without histological features of cancer were clas- sified as benign (five females and three males; aged from 21 to 74 yr old) and from eight patients with either histological malig- nancy or documented metastasis were classified as malignant (five females and three males; aged from 46 to 68 yr old). Tumor size ranged from 1 to 5 cm for adenomas and from 4 to 19 cm for carcinomas. Weiss score for ACCs was classified as 5.75 ± 0.25. Normal adrenal glands were obtained from eight subjects un- dergoing nephrectomy due to nonpapillous carcinoma of the kidney (three females and five males; aged from 39 to 72 yr old). The human adrenocortical carcinoma cell lines [NCI-H295R (ATCC CRL-2128; American Type Culture Collection, Manas- sas, VA) (21), SW-13 (ATCC CCL-105; American Type Culture Collection), and HAC-15 provided by Dr. W. E. Rainey (Medical College of Georgia, Augusta, GA) (22)] were included in this study. Informed consent for the analysis of all tissue samples was obtained from the patients in accordance with national ethical rules. The study was approved by the Ethical Committee of the University of Technology Dresden (Dresden, Germany).
Immunohistochemistry
Tissue samples were in 10% formalin (pH 7.4) and embedded in paraffin. Serial sections of 6 um were stained using an auto- mated immunostainer (BenchMark; Ventana, Tucson, AZ) ac- cording to the manufacturer’s protocols. Primary antibodies were mouse antihuman cluster of differentiation 14 (CD14) (di- lution 1:50, clone 5A3B11B5; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit antihuman TLR4 (dilution 1:50; Abcam, Cambridge, MA). The signal was amplified using the Ventana amplification kit and visualized using avidin-biotin labeling and 3,3’-diaminobenzidine. Slides were counterstained with hema-
toxylin. Staining with isotypic control antibodies (mouse IgG2, K; BD Bioscience/PharMingen, Oxford, UK) was performed to confirm staining specificity.
Cell culture and stimulation
Primary adrenocortical cells were isolated using enzymatic tissue digestion and were subsequently depleted of CD68-posi- tive macrophages with magnetic beads as previously described (23). NCI-H295R, HAC-15, and SW13 cells were grown as pre- viously described (24). Before incubation with TLR4 agonist, cells were grown without serum for 72 h. Thereafter cell were incubated with LPS (1 µg/ml) for 30 and 120 min.
Antibiotic selection and establishment of stable transfected NCI-H295R clones
NCI-H295R cells were transfected with expression plasmid (pDUO) encoding either TLR4-CD14 or with an empty plasmid negative control (MOCK; InvivoGen, San Diego, CA) using Li- pofectamine LTX PLUS transfection reagent (Invitrogen Life Technologies, Karlsruhe, Germany). Several stably TLR4- CD14-expressing clones of NCI-H295R cells were selected using blasticidin S (5 µg/ml; InvivoGen).
Cell viability and apoptosis
Cell proliferation/viability was analyzed using colorimetric Cell-Titer 96 Aqueous One solution cell proliferation assay (Pro- mega, Madison, WI). Apoptotic nuclei were identified using non- radioactive DeadEnd fluorometric terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end label- ing (TUNEL) system (Promega) according to the manufacturer’s protocol. Negative controls were performed without terminal deoxynucleotidyl transferase labeling. Quantification of apo- ptotic cells was accomplished using fluorescence microscopy by counting the total number of positively stained cells by the TUNEL assay from 1000 cells of each of three randomly chosen slide fields.
Statistics
All results are depicted as the mean ± SEM. Results were an- alyzed using one-way ANOVA followed by the Dunnett or Tukey posttests, using the GraphPad Prism version 4.02 (Graph- Pad Software, Inc., San Diego, CA). Differences with P < 0.05 were considered to be statistically significant.
For details concerning cell extracts and Western blot, electron microscopy and quantitative PCR, please refer to the Supple- mental Material, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org.
Results
Expression profile of TLR4, CD14, and myeloid differentiation protein-2 (MD2) in human adrenocortical tumors and ACC cell lines
Using real-time PCR, expression of TLR4, MD2, and CD14 mRNA was first examined in three ACC cell lines (SW-13, NCI-H295R, and HAC-15) and in human adre- nocortical cells in primary culture. Our results show that mRNA expression of TLR4, CD14, and MD2 was sig-
nificantly reduced in all adrenocortical cell lines tested compared with adrenocortical cells in primary culture (nHAC). TLR4 expression was significantly reduced in NCI-H295R (8-fold; P<0.001), HAC-15 (35-fold; P < 0.001), and SW-13 (>1000-fold; P < 0.001) (Fig. 1A). CD14 expression was significantly reduced in NCI- H295R (200-fold; P < 0.05), HAC-15 (82-fold; P < 0.05), and SW-13 (400-fold; P < 0.05) (Fig. 1B). MD2 expression was significantly reduced in two cell lines NCI- H295R (66-fold; P < 0.01) and SW-13 (14.5-fold; P < 0.01) (Fig. 1C). Because the adaptation to in vitro growth may alter the expression of many genes, the expression data were verified in tumor tissues obtained from patients with ACC and adenoma (ADN) compared with normal adrenal gland (nAdr). Our results show that the expres- sion of TLR4 and CD14 was significantly down-regulated in samples of ACCs and in ADNs. TLR4 was reduced in ACC(115-fold; P<0.001) and ADNs (36-fold; P<0.01) (Fig. 1D). Simultaneously, CD14 expression was found to be reduced in ACCs (38.43-fold; P < 0.001) and ADNs (22-fold; P < 0.05) (Fig. 1E). Finally, expression of MD2 was found to be significantly reduced in ACCs (13.8-fold; P < 0.01), whereas in ADN a strong tendency could be demonstrated (Fig. 1F).
Immunohistochemical analysis of TLR4 and CD14 expression in normal adrenal cortex and tumors
Immunohistochemical analysis of TLR4 and CD14 ex- pression patterns in normal adrenal gland, adrenal ade- nomas, and carcinomas are demonstrated in Fig. 2. These results (n = 8) confirm TLR4 expression in the human adrenal cortex (25); immunostaining was found in all three layers of the adult adrenal cortex. Furthermore, us- ing immunohistochemistry we show for the first time CD14 expression, which was localized at the membranes of adrenocortical cells of the inner cortical zones, namely zona fasciculata and zona reticularis (Fig. 2). Further- more, immunohistochemical analysis of adrenal tumors has shown that TLR4 and CD14 expression is abrogated in majority of ACC tissue samples, whereas in ADNs the expression of both proteins was partially preserved in both epithelial cancer cells and remnant normal tissue. In all tumor samples tested, single-cell staining of TLR4 and CD14 could be seen (Fig. 2, black arrows). Both isotopic- matched control antibody and staining without primary antibody were used as negative controls and showed no positive result.
Evaluation of the TLR4 signaling in human adrenocortical cells in primary culture and ACC cell lines
In THP-1 macrophages, TLR4 activation is known to trigger several conserved signaling pathways including the
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nuclear factor-KB (NF-KB), MAPK, or AKT. Since the pos- sible ligand of TLR4 in ACC is unknown LPS was used as the specific TLR4 agonist. Our results show that in con- trast to THP-1 cells, incubation of the adrenocortical car- cinoma cell line NCI-H295R with bacterial LPS, activates neither NF-KB nor MAPKs pathway. Similar results were obtained also in other ACC cell lines, SW-13 and HAC-15 cells (data not shown). Interestingly, the MAPK and AKT pathways were found to be activated already at basal con- ditions (Supplemental Data).
Reintroduction of TLR4-CD14 expression restores LPS-mediated NF-KB nuclear translocation
To elucidate the potential role of TLR4 and CD14 down-regulation in the process of adrenal tumorigenesis, we reintroduced TLR4 and CD14 expression in the NCI- H295R cells. Using TLR4-CD14 transfection and subse- quent antibiotic selection procedure, we generated several clones of NCI-H295R cells. Western blot analysis con- firmed the up-regulation of TLR4 and CD14 expression in three of seven randomly analyzed clones, which were fur- ther used for the experiments. A densitometrical analysis
of Western blot results obtained from the representative cell clone (Fig. 3A) showed that TLR4 expression in- creased by 1.52-fold and CD14 by 13.56-fold (Fig. 3B). The functionality of TLR4 and CD14 in the transfected NCI-H295R cell was evaluated by incubating these clones with of LPS (1 µg/ml) for 15 or 120 min, respectively, and analyzing NF-KB and MAPKs as well as AKT pathways. Our results show that enhanced expression of TLR4 and CD14 proteins sensitized NCI-H295R cells to LPS as shown by NF-KB nuclear translocation (Fig. 3C).
Induction of TLR4 and CD14 expression reduces basal and growth factor-induced viability of human adrenocortical carcinoma cells
TLR4 activation was found to regulate viability and proliferation in many carcinoma cell lines (26); therefore, we incubated serum-starved NCI-H295R cells expressing TLR4-CD14 or MOCK control cells with different con- centrations of LPS (10 up to 1000 ng/ml) for 24-72 h. Our results showed that endotoxin had no significant effect on cell viability between MOCK- and TLR4-CD14-trans- fected cells. However, viability of TLR-CD14-expressing
TLR4
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NCI-H295R cells was significantly reduced (83.99 ± 13.7%, P < 0.05), even at basal conditions to MOCK- transfected cells (Fig. 4A). We then evaluated the viability of these cells in response to insulin-transferrin-sodium se- lenite (ITS) supplement, which is known to promote pro- liferation of adrenocortical cells in vitro. In contrast to the MOCK-transfected cells, which showed a significant in- crease in cell number (134.2 ± 5.63%; P < 0.01) after ITS incubation, viability of NCI-H295R cells expressing TLR4-CD14 did not increase.
Restoration of TLR4 and CD14 expression increases apoptosis of human ACC cells
One possible explanation for the observed reduced growth and viability of TLR4- and CD14-expressing NCI- H295R cells could be an increased apoptotic or necrotic rate of these cells. Indeed, electron microscopical exami- nation of TLR4- and CD14-expressing NCI-H295R cells revealed an increased number of apoptotic cells (Fig. 4F) characterized by a high degree of cytoplasm and chroma- tin condensation and typical vacuolization compared with MOCK control cells (Fig. 4E). To quantify the apoptotic cells, the TUNEL assay was used (Fig. 4, C and D). Mi- croscopic examination and quantification of the positively stained cells showed a significantly higher number of ap- optotic cells in the NCI-H295R cells expressing TLR4-
CD14 than in MOCK transfected cells (17.99 ±1.8 vs. 9.33 ±0.33; P<0.01) (Fig. 4B).
Discussion
Steadily increasing evidence links expres- sion of TLR4, an innate immune receptor recognizing Gram-negative bacterial LPS, with the process of tumorigenesis (27). In the present study, we demonstrate an ab- rogation of TLR4 in adrenocortical tu- 2pm mors, which could explain an unrespon- siveness of adrenocortical carcinoma cells to LPS (13). Because this effect may result from the reduced expression of other molecules involved in LPS recog- nition, we examined the expression of 20 um, membrane glycoproteins CD14 and MD2 (28), which were also down-reg- ulated in the ACC cell lines compared with primary cultures of human adre- nocortical cells. To exclude the possi- bility that the reduced expression of TLR4 in human adrenocortical cells is the result of an adaptation to in vitro cell culture conditions, both human ACC and ADN tissue samples were analyzed. The expression of all three mole- cules involved in LPS recognition was significantly re- duced in the primary adrenal tumor samples consistent with the results obtained from the analysis of the adrenal carcinoma cell lines. The down-regulation of TLR4 ex- pression in tumor cells has been reported for some leuke- mic leukocyte populations, and this was linked to the general immunosuppressive status of this tumor (29). In accordance with our data, a transcriptome profiling of normal human adrenal cortex vs. ACCs revealed 91 genes that displayed at least 3-fold differential expres- sion including up-regulation of IGF-II and down-regu- lation of CD14 in ACC tissue samples (30).
To further elucidate the specific distribution of TLR4 and CD14 expression, we performed a comprehensive im- munohistochemical analysis of normal human adrenal gland and tumor tissues. In the normal adrenal, TLR4 was expressed in all three layers of the adrenal cortex, whereas CD14 membrane expression was found only in the inner cortical zones, namely fasiculata and reticularis. In con- trast to benign adenomas, no staining was shown in epi- thelial cells of ACCs.
Currently differentiation between malignant and be- nign adrenal tumors is based on the macroscopic analysis
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using criteria proposed by Weiss (20) including tumor weight, a high mitotic rate, tumor necroses, or atypical mitotic figures. Although histopathological examination still remains the gold standard in the diagnosis of adrenal malignancies, immunohistochemical examination of tu- mor biopsies (e.g. for higher expression of Ki67 or mu- tated p53, which characterize advanced ACC stages) can indicate a poor prognosis (31, 32). However, the expres- sion of the latter markers is not found in all ACC cases, necessitating the identification of additional markers. Our results show that an abrogation of TLR4 and CD14 ex- pression found in ACC samples but not in ADNs is a newly characterized feature of ACC and may be used as an ad- ditional tumor marker.
TLR4 has been found to play an important role in the process of tumorigenesis in many tissues. Thus, TLR4- mediated LPS ligation induced tumor evasion from im-
mune survivalence in murine tumor cell lines (17), and the overt expression of TLR4 in human lung cancer cells pro- moted the secretion of immunosup- pressive cytokines and resistance to TNF-a and TNF-related apoptosis-in- ducing ligand-induced apoptosis (33). These data suggest that activation of TLR4 in tumor cells may directly pro- mote cancer cell survival and chemore- sistance therefore facilitating tumor progression.
To verify the functionality of the newly restored proteins, we incubated THP-1, a macrophage cell line, MOCK- p transfected (control), and TLR4-CD14- expressing NCI-H295R cells with LPS t and analyzed the TLR-related activation of signaling pathways including NF-KB, activator protein-1, MAPKs, and AKT. We used LPS as an agonist of TLR4 be- p t cause its natural ligand in ACC is not yet known. Using Western blot, we p have shown that THP-1 macrophages incubated with LPS activate NF-KB, ac- t tivator protein-1, AKT, and MAPK pathways. In contrast, normal and MOCK-transfected NCI-H295R cells show basal activation of MAPK and AKT pathways, and incubation with LPS did not increase further the phos- phorylation of these kinases. An in- creased activation of the AKT pathway in ACCs has been reported previously, suggesting a critical role of this kinase in the adrenal tumorigenesis (34). LPS activation, however, was capable of inducing NF-KB nu- clear translocation after 30 and 120 min in TLR4- and CD14-expressing NCI-H295R cells; this verifies the func- tionality of both proteins and additionally shows that TLR4 signaling is intact in these cells.
To date, little is known about the function of TLR ex- pressed in tumor cells. On the one hand, a local and sys- temic delivery of TLR4 ligands had anticancer properties. On the other hand, TLR4 administration in a variety of tumor cell lines let to an increased survival and prolifer- ation in vitro. Therefore, to elucidate the potential func- tion of TLR4 in the process of adrenal cortex tumorigen- esis, both NCI-H295R cell proliferation and apoptosis have been compared in MOCK- and TLR4-CD14-trans- fected cells. Our results show that ITS, cell growth-en- hancing media supplement, induced a significant prolif-
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eration of MOCK-transfected NCI-H295R cells but had no effect on TLR4- and CD14-expressing cells; in fact, the proliferation of TLR4- and CD14-expressing NCI- H295R cells was also significantly decreased in basal conditions. To find a possible explanation for this ob- servation, we analyzed the ultrastructure of both MOCK- transfected and TLR4-CD14-expressing NCI-H295R cells. Electron microscopic examination revealed an in- creased apoptosis in TLR4- and CD14-expressing cells as compared with MOCK control. Hence, our results suggest that restoration of TLR4 and CD14 expression in NCI- H295R cells may decrease rather than promote viability of NCI-H295R cells, even in the absence of LPS. To quantify the number of apoptotic cells, we used the TUNEL assay, which revealed the doubling of apoptotic cells in TLR4- CD14-expressing cells compared with MOCK-trans- fected cells. Interestingly, incubation with LPS did not further increase the number of apoptotic cells, suggest- ing that other agonists of TLR4 may be responsible for this effect.
One of the potential TLR4 ligands that is indeed over- expressed in many ACC is heat shock protein 60 (35, 36). Heat shock protein 60 is known to induce apoptosis when released from the mitochondrial compartment of injured cells via caspase-3-dependent mechanism in many cell types including cardiomyocytes or endothelial cells (37). Moreover, a direct proapoptotic effect of TLR ligands has already been documented in the case of TLR4, TLR3, and TLR9 ligands in various carcinoma cell lines and in vivo studies (38-40). Furthermore, in the pituitary carcinoma cell line AtT20 and in the TLR4-positive pituitary ade- noma primary cell cultures, activation of TLR4 by LPS has been shown to induce IL-6 production and simultaneously dose-dependently reduce cell growth (41).
In conclusion, our results demonstrate the significant reduction of TLR4 and CD14 expression and signaling in malignant but not benign adrenal tumors, which is a novel feature of adrenocortical tumors and may be used as a diagnostic marker. Moreover, an immune-mediated up- regulation of TLR4 expression and signaling in ACC cells
may provide a novel therapeutic strategy for immunother- apy of adrenal cancer.
Acknowledgments
We are grateful to Professor Volker Janitzky and Dr. Torsten Weirich for generously providing tissue specimens. We thank Professor William Rainey and Dr. Jeniel Parmar for kindly pro- viding HAC-15 cells. We greatly appreciate the help from Silke Zeugner for performing the excellent immunohistochemical staining and Uta Buro for performing real-time PCR.
Address all correspondence and requests for reprints to: Stefan R. Bornstein, Department of Medicine III, Carl Gustav Carus Medical School, University of Technology Dresden, Fetscherstrasse 74, 01307 Dresden, Germany. E-mail: stefan. bornstein@uniklinikum-dresden.de.
This work was supported by Grants 2006.021.3 from the Sander Foundation (to S.R.B.) and ZA 243 11-4 (to K.Z.) from the Deutsche Forschungsgemeinschaft.
Disclosure Summary: The authors reported no potential con- flict of interest.
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