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Androgen blockade enhances response to melanoma vaccine1
Journal of Surgical Research 110, 393–398 (2003) doi:10.1006/jsre.2002.6608
Androgen Blockade Enhances Response to Melanoma Vaccine 1 Eddy C. Hsueh, M.D.,* ,2 Rishab K. Gupta, Ph.D.,* Alan Lefor, M.D.,† Gary Reyzin, B.S.,* Wei Ye, M.S.,* and Donald L. Morton, M.D.* *Department of Surgical Oncology, John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, California 90404 and †Cedars Sinai Medical Center, Los Angeles, California Submitted for publication October 11, 2002
Background. Because preclinical studies suggest an interaction between androgens and the immune system, we used a murine model to determine whether androgen blockade with flutamide might enhance the immunogenicity of an irradiated melanoma cell vaccine. Materials and methods. Forty C57BL/6 male mice were randomly assigned to four treatment groups: flutamide ⴙ RPMI (Group A), flutamide ⴙ irradiated B16 murine melanoma cells (Group B), placebo ⴙ RPMI (Group C), and placebo ⴙ irradiated B16 cells (Group D). Splenocyte proliferation and secretion of interleukin-2 and interferon-gamma were assayed after coculturing splenocytes with irradiated B16 cells. Antibody-dependent cellular cytotoxicity (ADCC) against B16 cells was determined using peripheral blood lymphocytes. To examine the effect of treatment on tumor growth, a second set of 40 mice assigned to Groups A, B, C, and D underwent tumor challenge 7 days after the last treatment. Results. Splenocyte proliferation was significantly higher in the two groups receiving flutamide at 50 mg/kg ⴛ 7 days (29% in Groups A and B vs 3% in Group C and 7% in Group D). Secretion of interferon was significantly higher in mice receiving flutamide ⴙ irradiated B16 cells (15.2 pg/ml in Group B vs 0, 1.7, and 4 pg/ml in Groups A, C, and D, respectively; P ⴝ 0.0024). Differences in interleukin secretion were not significant. ADCC was 26% in Group B vs 15, 8, and 22% in Groups A, C, and D, respectively (P ⴝ 0.0001). In the tumor challenge experiment, the rate of survival was 1 This work was supported in part by a Young Investigator Award from the American Society of Clinical Oncology (to Dr. Hsueh). Presented at the annual meeting of the American Society of Clinical Oncology, San Francisco, May 12–15, 2001. 2 To whom correspondence and reprint requests should be addressed at John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404. Fax: 1-310-449-5261. E-mail: hsuehe@ jwci.org.
10% higher in mice receiving irradiated B16 ⴙ flutamide than in mice receiving irradiated B16 alone. Conclusion. Flutamide can enhance immune responses to an irradiated whole-cell melanoma vaccine. A clinical study of immunotherapeutic androgen blockade is warranted. © 2003 Elsevier Science (USA) Key Words: immunotherapy; melanoma; androgen; flutamide. INTRODUCTION
In the United States, the incidence of melanoma is increasing faster than that of any other malignant neoplasm, except lung cancer in women [1]. Because of its resistance to chemotherapy and radiation therapy, melanoma has been treated primarily by surgery. At the John Wayne Cancer Institute, our investigations of active specific immunotherapy using a polyvalent whole-cell melanoma vaccine have shown promising clinical efficacy in surgical and nonsurgical patients with metastatic melanoma [2, 3]. However, neither nonspecific immunostimulation with cytokines nor specific immunostimulation with defined antigens elicits an immune response in all patients. Failure of the host to mount a clinically effective antitumor immune response is most likely due to ineffective presentation of tumor antigens to the host’s immune system. Although various adjuvants can enhance antigen presentation, there is no consensus regarding the ideal adjuvant to optimize stimulation by immunotherapeutic agents. Adjuvants currently employed in melanoma immunotherapy, e.g., QS-21, Bacillus Calmette-Gue´rin (BCG), and keyhole limpet hemocyanin (KLH), enhance the local presentation of tumor antigens to the host. Successful antigen presentation may require systemic manipulation of the host’s immune system. This suggests a hormonal mechanism.
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Melanoma is an immunogenic tumor. Clinical observations in melanoma patients strongly indicate an interaction between hormonal and immune systems. A clinically relevant hormone-driven response to melanoma is suggested by multivariate analyses that consistently report a gender-based difference in long-term survival [4 – 8]. In early-stage melanoma, these studies have found that females have a 10 to 15% higher rate of 10-year survival [4, 6, 7]. In metastatic melanoma, the clinical efficacy of chemotherapy is reportedly higher in female patients [5, 8]. The interaction between gonadal hormones and immune function may provide the key for systemic manipulation of the host’s immune system to react to the locally delivered tumor antigen. Our group has demonstrated the clinical importance of specific antibody responses in predicting the survival of patients receiving active specific immunotherapy for distant metastatic melanoma [9]. We have also demonstrated the prognostic significance of complementdependent cytotoxicity (CDC) in patients receiving a polyvalent whole-cell melanoma vaccine [10]. The importance of antibody-dependent cellular cytotoxicity (ADCC) in active specific immunotherapy has been demonstrated by other investigators [11]. Preclinical data show that androgen blockade enhances B-cell lymphopoiesis in bone marrow and spleen, cytokine secretion in the spleen, and the proliferation of splenocytes [12–17]. Recent findings in a trauma-hemorrhage model suggest that antigenic challenge in the presence of androgen blockade with flutamide increases the production of interleukins 1 and 2 and the proliferation of spleen cells [18]. Thus, we hypothesized that androgen blockade might enhance the immune response to active specific immunization in a syngeneic murine melanoma model. METHODOLOGY
Animals Seven- to 8-week-old C57BL/6 male mice were obtained from Harlan Laboratories, Indianapolis, Indiana. All mice were housed and fed under conventional conditions. All animal experiments were carried out under conditions that complied with regulations stipulated by the animal subjects committee and guidelines set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). The protocol was approved by the Institutional Animal Care & Use Committee (IACUC) of Cedars-Sinai Medical Center.
Experimental Design Dose escalation study. Flutamide is potent nonsteroidal antiandrogen widely used for treatment of patients with metastatic prostate cancer. It acts by inhibiting the binding of androgens to their receptors in the target cell nucleus. Because the effect of androgen blockade on host response to tumor vaccine has not been tested before, a dose-finding study was necessary prior the experimental treatment phase. The selection of flutamide doses was based on studies in a trauma-hemorrhage mouse model [18].
Twenty-five mice in randomly assigned groups of five received seven daily subcutaneous injections of flutamide at doses of 0, 12.5, 25, 50, or 100 mg/kg in alternate hindquarters (days 1 to 7). After the last flutamide injection (day 7), all 25 mice were immunized intradermally with 1 ⫻ 10 6 irradiated B16-F1 cells. Blood was obtained from each animal by tail-tip amputation before flutamide treatment (day 1) and 1 week after vaccine treatment (day 14). On day 14, animals were anesthetized, blood was collected via inferior vena cava cannulation, and spleens were harvested. The optimal dose of flutamide was then determined. Experimental phase. Forty mice were randomized into four treatment groups of 10 animals each. Groups B and D (vaccine mice) were immunized on day 7 with 1 ⫻ 10 6 irradiated B16-F1 cells; Groups A and C (control mice) were immunized intradermally on day 7 with an equal volume of RPMI-1640. For 7 days before immunization (days 1–7), Groups A and B received daily subcutaneous injections of flutamide at the dose determined in the dose-escalation study; Groups C and D received an equal volume of 1,2-propane-diol (Sigma; St. Louis, MO), which served as placebo. Blood was collected before flutamide treatment (day 1) and on day 14 (7 days after immunization). These blood specimens were used to assess ADCC. After blood collection on day 14, mice were anesthetized and sacrificed for spleen harvest. To determine the effect of androgen blockade on the prevention of tumor cell growth, an additional 40 mice were randomized to Groups A, B, C, and D. On day 14, these mice were challenged with a dose of 1 ⫻ 10 5 nonirradiated live B16-F1 tumor cells injected subcutaneously in the hindquarters. Survival was evaluated, and moribund mice were euthanized by CO 2. Sixty-five days after live tumor-cell injection, the surviving animals were euthanized.
Preparation of B16 Cells B16-F1 cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 g/ml streptomycin. To prepare whole-cell vaccine, B16-F1 cells were irradiated with 10,000 rad and stored in liquid nitrogen until use. Prior to administration, the cells were thawed and washed three times with RPMI-1640; viable cells were counted with a cell counter.
Splenocyte Preparation After CO 2 euthanasia, the spleen of each mouse was removed under sterile conditions and placed in a petri dish containing cold (4°C) phosphate-buffered saline solution. The spleen was minced and vortexed, and red cells were lysed with RBC lysis solution (Gentra Systems, Minneapolis, MN). The splenocytes were then counted and aliquoted.
Cytokine Release Assay Splenocytes were incubated for 48 h at 37°C in 5% CO 2 at 90% humidity, with or without 1 ⫻ 10 6 irradiated B16-F1 cells in splenocyte-cell ratios of 1:1, 1:5, and 5:1. The cell suspension was then centrifuged and the supernatant harvested and cryopreserved for cytokine secretion assays. The cytokine assay was determined by sandwich enzyme-linked immunosorbent assay (ELISA) techniques using monoclonal antibodies to IL2 and IFN-␥ (PharMingen, San Diego, CA). Briefly, anticytokine capture antibody was adsorbed to each well of a 96-well polypropylene ELISA plate overnight at 4°C. The ELISA plate was then blocked with mouse serum, and the test supernatant at dilutions of 1:10, 1:20, 1:40, 1:80, and 1:160 was loaded into the well. An equal volume of standard (recombinant mouse IL2 or IFN-␥, PharMingen) also was loaded into each plate. After the ELISA plate was incubated overnight at 4°C, biotinylated anticytokine detection antibody was added to each well. The plate was then incubated at 22°C for 45 min. Substrate was added to each
HSUEH ET AL.: ANDROGEN BLOCKADE TO ENHANCE MELANOMA VACCINE
FIG. 1.
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Splenocyte secretion of cytokines IL2 and IFN-␥ in response to titration of flutamide dose.
well and incubated at room temperature for color development. The absorbance was measured at 405 nm with a microplate reader (Titertek Multiscan, Dynatech, Alexandria, VA). The assays were performed in triplicate and repeated twice.
Splenocyte Proliferation Assay Splenocyte proliferation in response to vaccine immunization was assessed by incubating the splenocytes for 72 h at 37°C in 5% CO 2 at 90% humidity, with or without irradiated B16-F1 cells. The extent of proliferation was measured by tetrazolium-based colorimetric method using the CellTiter 96 AQ ueous Non-Radioactive Cell Proliferation Assay Kit from Promega (Madison, WI) according to the manufacturer’s protocol.
Antibody-Dependent Cell-Mediated Cytotoxicity ADCC was performed on peripheral blood obtained at day 7. Mononuclear cells were isolated from whole blood by Ficoll gradient. The mononuclear cells were then harvested and washed with RPMI1640. The viable cells were counted in 0.2% trypan blue in PBS. B16-F1 cells served as target cells. The target cells at 2 ⫻ 10 5 cell/ml were incubated with heat-inactivated test serum at 4°C for 1 h. Mononuclear effector cells were then added at effector/target-cell ratios between 5:1 and 50:1. The final mixture was incubated at 37°C for 4 h. ADCC was determined by amount of LDH over background and controls as measured by the Cytotox assay kit (Promega). Controls were PBL, irradiated B16 cells, and RPMI/FBS.
Statistical Analysis Results were expressed as mean values ⫾ standard error of measurement (SEM). Comparison among the groups was performed by Kruskal-Wallis Test and Wilcoxon signed rank sum test. A P value ⬍0.05 was considered significant.
RESULTS
Dose Escalation Study Seven days of flutamide administration followed by injection of irradiated B16 tumor vaccine produced a dose-dependent increase in stimulated splenocyte IFN-␥ secretion (Fig. 1). Splenocyte secretion of IFN-␥ was highest in mice receiving 100 mg/kg/day of flutamide subcutaneously, whereas splenocyte secretion of IL2 was highest in mice receiving 50 mg/kg/day of flutamide (Fig. 1). After challenge with irradiated B16 tumor vaccine, splenocyte proliferation was highest in mice that had received 50 mg/kg/day of flutamide. Splenocyte proliferation increased 0% for control and 12.5-mg/kg flutamide groups, 13% ⫾ 0.7% for the 25mg/kg flutamide group, 24% ⫾ 1.2% for the 50-mg/kg group, and 15% ⫾ 0.8% for the 100-mg/kg group. Thus, a flutamide dosage of 50 mg/kg for 7 days prior to immunization was selected for subsequent evaluation in Groups A, B, C, and D. Splenocyte Proliferation Study Splenocyte proliferation was significantly higher (P ⫽ 0.0031) in the two flutamide groups (Group A: 29% ⫾ 1.5% and Group B: 29% ⫾ 1.5%) than in the two control groups (Group C: 3% ⫾ 0.2% and Group D: 7% ⫾ 0.4%), but there was no significant difference for mice receiving flutamide plus RPMI (Group A) vs flu-
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FIG. 2. Splenocyte secretion of IL2 and IFN-␥ in mice that were treated with or without flutamide and then challenged with irradiated B16 tumor vaccine or media alone. Splenocyte secretion of IFN-␥ was significantly higher in mice treated with flutamide plus irradiated B16 cells (Group B) than in the other three groups (P ⫽ 0.0024). Splenocyte secretion of IL2 also was highest in Group B but was not significantly different among the four groups (P ⫽ 0.2047).
tamide plus B16 (Group B). Although splenocyte proliferation was higher in mice receiving B16 alone (Group D) than in mice receiving placebo plus RPMI (Group C), this difference was not significant. Cytokine Secretion Assay Splenocyte secretion of IFN-␥ was significantly higher in mice treated with flutamide plus B16 cells (Group B) than in the other three groups (P ⫽ 0.0024) (Fig. 2). Splenocyte secretion of IL2 was also highest in Group B but did not differ significantly among the four groups (P ⫽ 0.2047) (Fig. 2). ADCC ADCC against B16 cells was significantly different (P ⫽ 0.0001) among the four groups. At a 5:1 effectortarget ratio, ADCC was 26% ⫾ 1.3% in mice receiving flutamide plus irradiated B16 (Group B), compared with 22% ⫾ 1.1% in mice receiving vehicle plus B16 (Group D); this difference approached statistical significance (P ⫽ 0.0746). The ADCC of Group B was significantly higher (P ⬍ 0.05) than ADCC values for Groups A (15% ⫾ 0.8%) and C (8% ⫾ 0.4%).
Tumor Growth Prevention Study At day 65, the survival rate was highest for the group of mice treated with irradiated B16 plus flutamide (Fig. 3). When the four mice left in this group were euthanized on day 65, there was no evidence of tumor on necropsy. In contrast, two of the three surviving mice in the irradiated B16 alone group were tumor-free on necropsy, and none of the surviving mice in the other two control groups were tumor-free on necropsy. DISCUSSION
In this murine study, androgen blockade enhanced immune responses (splenocyte proliferation, splenocytestimulated secretion of IFN-␥, and ADCC) to subsequent administration of an irradiated whole-cell melanoma vaccine. Other preclinical studies have examined the interaction between androgen and the immune system. In a murine study, androgen administration caused atrophy of the thymus and depression of lymphopoiesis [12], whereas castration increased the weight of the thymus and spleen [13]. Castrated male mice reportedly have a significant increase in splenic B lymphocytes compared with sham-operated male mice
HSUEH ET AL.: ANDROGEN BLOCKADE TO ENHANCE MELANOMA VACCINE
FIG. 3. Tumor growth prevention study. Mice were randomly assigned to four groups (n ⫽ 10 in each group). Each group underwent a 7-day treatment regimen that was followed by injection of vaccine and 7 days later by injection of live nonirradiated B16 cells. One group received 50 mg/kg/day flutamide and irradiated B16 tu). The second group received 50 mor vaccine (thick black line mg/kg/day flutamide and RPMI (vaccine control) (thin black line O). The third group received vehicle (1,2-propane-diol) and irradiated B16 tumor vaccine (thick gray line ). The fourth group received vehicle (1,2-propane-diol) and RPMI (thin gray line ).
[14]. Similarly, increased B-lymphocyte lymphopoiesis has been demonstrated in the bone marrow of castrated male mice [15]. A study using androgeninsensitive mice found a significant increase of B-cell precursors (CD45R⫹, IgM⫺) and mature B cells (sIgM⫹) in the bone marrow and a significant increase of mature B cells in the spleen [16]. Androgen also inhibited in vitro B-cell precursor expansion in the presence of stromal cells. Other murine studies have reported a reduction of bone marrow B-lymphocyte formation after androgen administration [17], and an increase in the release of IL2 and IFN-␥ by peripheral T cells in response to mitogen (Con A) after castration [13]. In the clinical setting, the effects of androgen on the immune system have been evaluated in patients undergoing androgen replacement therapy. In patients with Klinefelter’s syndrome, which is characterized by gynecomastia and underandrogenization [19], a decrease in the number of CD4⫹ cells and the CD4⫹/ CD8⫹ ratio as well as an increase in the number of CD8⫹ cells was observed following androgen replacement therapy [20, 21]. The decrease in CD4⫹/CD8⫹ ratio was also observed in rheumatoid arthritis patients receiving androgen replacement therapy [22]. Interestingly, total lymphocyte counts, CD4⫹ T cell number, and CD4⫹/CD8⫹ ratio were higher in patients with Klinefelter’s syndrome than in agematched healthy controls [20]. The effects of androgen on lymphocytes may be mediated by other immunoreactive signals. Expression of the androgen receptor (AR) could not be demonstrated in T lymphocytes, peripheral B cells, or in bone marrow
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B-cell precursors [23, 24]. We were unable to detect AR expression by immunohistochemical staining of 20 human melanoma biopsy specimens or by flow cytometry on B16 murine melanoma cells (E. Hsueh, unpublished data). However, other investigators have demonstrated AR expression in thymocytes [25]. Furthermore, action of androgen on lymphocytes may be mediated by the immune reactive signals from epithelial cells or macrophages [26, 27]. The results of our murine study are consistent with the reported effects of androgen blockade. The results obtained in the splenocyte proliferation assay indicated a generalized immunologic enhancement with androgen blockade, whereas the results obtained with IFN-␥ secretion assay suggested a specific immune enhancement to the immunizing agent (irradiated B16 tumor vaccine) after androgen blockade. A flutamideinduced specific immune enhancement to the immunizing agent was also observed in the ADCC experiment. The ability of androgen blockade to enhance host response to tumor vaccination is novel and, to our knowledge, has not been tested before. Our tumor growth prevention study demonstrated that androgen blockade with flutamide had a mild-tomoderate protective effect. This is not surprising because B16-F1 is a poorly immunogenic tumor whose growth may not be obliterated solely by immune manipulations. However, even a delay in growth is significant. In our study, all mice were male. The level of androgen in female mice might not have been high enough to yield a similar response to blockade with flutamide, and further evaluation is necessary to determine the effects of androgen blockade in females receiving immunotherapy. Nonetheless, the implications of the current study are potentially significant for males with cancer. The clinical combination of androgen blockade with immunotherapy might be especially important for patients with metastatic prostate cancer, in whom androgen blockade also has significant therapeutic role. In conclusion, androgen blockade can enhance immune responsiveness to tumor vaccine immunotherapy in this murine melanoma model. Our findings suggest that the clinical application of this combination might have an additive and synergistic effect on tumor killing. This attractive possibility merits clinical investigation of androgen blockade as adjuvant in trials of immunotherapy for advanced cancer. REFERENCES 1.
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Androgen Blockade Enhances Response to Melanoma Vaccine 1 Eddy C. Hsueh, M.D.,* ,2 Rishab K. Gupta, Ph.D.,* Alan Lefor, M.D.,† Gary Reyzin, B.S.,* Wei Ye, M.S.,* and Donald L. Morton, M.D.* *Department of Surgical Oncology, John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, California 90404 and †Cedars Sinai Medical Center, Los Angeles, California Submitted for publication October 11, 2002
Background. Because preclinical studies suggest an interaction between androgens and the immune system, we used a murine model to determine whether androgen blockade with flutamide might enhance the immunogenicity of an irradiated melanoma cell vaccine. Materials and methods. Forty C57BL/6 male mice were randomly assigned to four treatment groups: flutamide ⴙ RPMI (Group A), flutamide ⴙ irradiated B16 murine melanoma cells (Group B), placebo ⴙ RPMI (Group C), and placebo ⴙ irradiated B16 cells (Group D). Splenocyte proliferation and secretion of interleukin-2 and interferon-gamma were assayed after coculturing splenocytes with irradiated B16 cells. Antibody-dependent cellular cytotoxicity (ADCC) against B16 cells was determined using peripheral blood lymphocytes. To examine the effect of treatment on tumor growth, a second set of 40 mice assigned to Groups A, B, C, and D underwent tumor challenge 7 days after the last treatment. Results. Splenocyte proliferation was significantly higher in the two groups receiving flutamide at 50 mg/kg ⴛ 7 days (29% in Groups A and B vs 3% in Group C and 7% in Group D). Secretion of interferon was significantly higher in mice receiving flutamide ⴙ irradiated B16 cells (15.2 pg/ml in Group B vs 0, 1.7, and 4 pg/ml in Groups A, C, and D, respectively; P ⴝ 0.0024). Differences in interleukin secretion were not significant. ADCC was 26% in Group B vs 15, 8, and 22% in Groups A, C, and D, respectively (P ⴝ 0.0001). In the tumor challenge experiment, the rate of survival was 1 This work was supported in part by a Young Investigator Award from the American Society of Clinical Oncology (to Dr. Hsueh). Presented at the annual meeting of the American Society of Clinical Oncology, San Francisco, May 12–15, 2001. 2 To whom correspondence and reprint requests should be addressed at John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404. Fax: 1-310-449-5261. E-mail: hsuehe@ jwci.org.
10% higher in mice receiving irradiated B16 ⴙ flutamide than in mice receiving irradiated B16 alone. Conclusion. Flutamide can enhance immune responses to an irradiated whole-cell melanoma vaccine. A clinical study of immunotherapeutic androgen blockade is warranted. © 2003 Elsevier Science (USA) Key Words: immunotherapy; melanoma; androgen; flutamide. INTRODUCTION
In the United States, the incidence of melanoma is increasing faster than that of any other malignant neoplasm, except lung cancer in women [1]. Because of its resistance to chemotherapy and radiation therapy, melanoma has been treated primarily by surgery. At the John Wayne Cancer Institute, our investigations of active specific immunotherapy using a polyvalent whole-cell melanoma vaccine have shown promising clinical efficacy in surgical and nonsurgical patients with metastatic melanoma [2, 3]. However, neither nonspecific immunostimulation with cytokines nor specific immunostimulation with defined antigens elicits an immune response in all patients. Failure of the host to mount a clinically effective antitumor immune response is most likely due to ineffective presentation of tumor antigens to the host’s immune system. Although various adjuvants can enhance antigen presentation, there is no consensus regarding the ideal adjuvant to optimize stimulation by immunotherapeutic agents. Adjuvants currently employed in melanoma immunotherapy, e.g., QS-21, Bacillus Calmette-Gue´rin (BCG), and keyhole limpet hemocyanin (KLH), enhance the local presentation of tumor antigens to the host. Successful antigen presentation may require systemic manipulation of the host’s immune system. This suggests a hormonal mechanism.
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0022-4804/03 $35.00 © 2003 Elsevier Science (USA) All rights reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 110, NO. 2, APRIL 2003
Melanoma is an immunogenic tumor. Clinical observations in melanoma patients strongly indicate an interaction between hormonal and immune systems. A clinically relevant hormone-driven response to melanoma is suggested by multivariate analyses that consistently report a gender-based difference in long-term survival [4 – 8]. In early-stage melanoma, these studies have found that females have a 10 to 15% higher rate of 10-year survival [4, 6, 7]. In metastatic melanoma, the clinical efficacy of chemotherapy is reportedly higher in female patients [5, 8]. The interaction between gonadal hormones and immune function may provide the key for systemic manipulation of the host’s immune system to react to the locally delivered tumor antigen. Our group has demonstrated the clinical importance of specific antibody responses in predicting the survival of patients receiving active specific immunotherapy for distant metastatic melanoma [9]. We have also demonstrated the prognostic significance of complementdependent cytotoxicity (CDC) in patients receiving a polyvalent whole-cell melanoma vaccine [10]. The importance of antibody-dependent cellular cytotoxicity (ADCC) in active specific immunotherapy has been demonstrated by other investigators [11]. Preclinical data show that androgen blockade enhances B-cell lymphopoiesis in bone marrow and spleen, cytokine secretion in the spleen, and the proliferation of splenocytes [12–17]. Recent findings in a trauma-hemorrhage model suggest that antigenic challenge in the presence of androgen blockade with flutamide increases the production of interleukins 1 and 2 and the proliferation of spleen cells [18]. Thus, we hypothesized that androgen blockade might enhance the immune response to active specific immunization in a syngeneic murine melanoma model. METHODOLOGY
Animals Seven- to 8-week-old C57BL/6 male mice were obtained from Harlan Laboratories, Indianapolis, Indiana. All mice were housed and fed under conventional conditions. All animal experiments were carried out under conditions that complied with regulations stipulated by the animal subjects committee and guidelines set forth in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). The protocol was approved by the Institutional Animal Care & Use Committee (IACUC) of Cedars-Sinai Medical Center.
Experimental Design Dose escalation study. Flutamide is potent nonsteroidal antiandrogen widely used for treatment of patients with metastatic prostate cancer. It acts by inhibiting the binding of androgens to their receptors in the target cell nucleus. Because the effect of androgen blockade on host response to tumor vaccine has not been tested before, a dose-finding study was necessary prior the experimental treatment phase. The selection of flutamide doses was based on studies in a trauma-hemorrhage mouse model [18].
Twenty-five mice in randomly assigned groups of five received seven daily subcutaneous injections of flutamide at doses of 0, 12.5, 25, 50, or 100 mg/kg in alternate hindquarters (days 1 to 7). After the last flutamide injection (day 7), all 25 mice were immunized intradermally with 1 ⫻ 10 6 irradiated B16-F1 cells. Blood was obtained from each animal by tail-tip amputation before flutamide treatment (day 1) and 1 week after vaccine treatment (day 14). On day 14, animals were anesthetized, blood was collected via inferior vena cava cannulation, and spleens were harvested. The optimal dose of flutamide was then determined. Experimental phase. Forty mice were randomized into four treatment groups of 10 animals each. Groups B and D (vaccine mice) were immunized on day 7 with 1 ⫻ 10 6 irradiated B16-F1 cells; Groups A and C (control mice) were immunized intradermally on day 7 with an equal volume of RPMI-1640. For 7 days before immunization (days 1–7), Groups A and B received daily subcutaneous injections of flutamide at the dose determined in the dose-escalation study; Groups C and D received an equal volume of 1,2-propane-diol (Sigma; St. Louis, MO), which served as placebo. Blood was collected before flutamide treatment (day 1) and on day 14 (7 days after immunization). These blood specimens were used to assess ADCC. After blood collection on day 14, mice were anesthetized and sacrificed for spleen harvest. To determine the effect of androgen blockade on the prevention of tumor cell growth, an additional 40 mice were randomized to Groups A, B, C, and D. On day 14, these mice were challenged with a dose of 1 ⫻ 10 5 nonirradiated live B16-F1 tumor cells injected subcutaneously in the hindquarters. Survival was evaluated, and moribund mice were euthanized by CO 2. Sixty-five days after live tumor-cell injection, the surviving animals were euthanized.
Preparation of B16 Cells B16-F1 cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 g/ml streptomycin. To prepare whole-cell vaccine, B16-F1 cells were irradiated with 10,000 rad and stored in liquid nitrogen until use. Prior to administration, the cells were thawed and washed three times with RPMI-1640; viable cells were counted with a cell counter.
Splenocyte Preparation After CO 2 euthanasia, the spleen of each mouse was removed under sterile conditions and placed in a petri dish containing cold (4°C) phosphate-buffered saline solution. The spleen was minced and vortexed, and red cells were lysed with RBC lysis solution (Gentra Systems, Minneapolis, MN). The splenocytes were then counted and aliquoted.
Cytokine Release Assay Splenocytes were incubated for 48 h at 37°C in 5% CO 2 at 90% humidity, with or without 1 ⫻ 10 6 irradiated B16-F1 cells in splenocyte-cell ratios of 1:1, 1:5, and 5:1. The cell suspension was then centrifuged and the supernatant harvested and cryopreserved for cytokine secretion assays. The cytokine assay was determined by sandwich enzyme-linked immunosorbent assay (ELISA) techniques using monoclonal antibodies to IL2 and IFN-␥ (PharMingen, San Diego, CA). Briefly, anticytokine capture antibody was adsorbed to each well of a 96-well polypropylene ELISA plate overnight at 4°C. The ELISA plate was then blocked with mouse serum, and the test supernatant at dilutions of 1:10, 1:20, 1:40, 1:80, and 1:160 was loaded into the well. An equal volume of standard (recombinant mouse IL2 or IFN-␥, PharMingen) also was loaded into each plate. After the ELISA plate was incubated overnight at 4°C, biotinylated anticytokine detection antibody was added to each well. The plate was then incubated at 22°C for 45 min. Substrate was added to each
HSUEH ET AL.: ANDROGEN BLOCKADE TO ENHANCE MELANOMA VACCINE
FIG. 1.
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Splenocyte secretion of cytokines IL2 and IFN-␥ in response to titration of flutamide dose.
well and incubated at room temperature for color development. The absorbance was measured at 405 nm with a microplate reader (Titertek Multiscan, Dynatech, Alexandria, VA). The assays were performed in triplicate and repeated twice.
Splenocyte Proliferation Assay Splenocyte proliferation in response to vaccine immunization was assessed by incubating the splenocytes for 72 h at 37°C in 5% CO 2 at 90% humidity, with or without irradiated B16-F1 cells. The extent of proliferation was measured by tetrazolium-based colorimetric method using the CellTiter 96 AQ ueous Non-Radioactive Cell Proliferation Assay Kit from Promega (Madison, WI) according to the manufacturer’s protocol.
Antibody-Dependent Cell-Mediated Cytotoxicity ADCC was performed on peripheral blood obtained at day 7. Mononuclear cells were isolated from whole blood by Ficoll gradient. The mononuclear cells were then harvested and washed with RPMI1640. The viable cells were counted in 0.2% trypan blue in PBS. B16-F1 cells served as target cells. The target cells at 2 ⫻ 10 5 cell/ml were incubated with heat-inactivated test serum at 4°C for 1 h. Mononuclear effector cells were then added at effector/target-cell ratios between 5:1 and 50:1. The final mixture was incubated at 37°C for 4 h. ADCC was determined by amount of LDH over background and controls as measured by the Cytotox assay kit (Promega). Controls were PBL, irradiated B16 cells, and RPMI/FBS.
Statistical Analysis Results were expressed as mean values ⫾ standard error of measurement (SEM). Comparison among the groups was performed by Kruskal-Wallis Test and Wilcoxon signed rank sum test. A P value ⬍0.05 was considered significant.
RESULTS
Dose Escalation Study Seven days of flutamide administration followed by injection of irradiated B16 tumor vaccine produced a dose-dependent increase in stimulated splenocyte IFN-␥ secretion (Fig. 1). Splenocyte secretion of IFN-␥ was highest in mice receiving 100 mg/kg/day of flutamide subcutaneously, whereas splenocyte secretion of IL2 was highest in mice receiving 50 mg/kg/day of flutamide (Fig. 1). After challenge with irradiated B16 tumor vaccine, splenocyte proliferation was highest in mice that had received 50 mg/kg/day of flutamide. Splenocyte proliferation increased 0% for control and 12.5-mg/kg flutamide groups, 13% ⫾ 0.7% for the 25mg/kg flutamide group, 24% ⫾ 1.2% for the 50-mg/kg group, and 15% ⫾ 0.8% for the 100-mg/kg group. Thus, a flutamide dosage of 50 mg/kg for 7 days prior to immunization was selected for subsequent evaluation in Groups A, B, C, and D. Splenocyte Proliferation Study Splenocyte proliferation was significantly higher (P ⫽ 0.0031) in the two flutamide groups (Group A: 29% ⫾ 1.5% and Group B: 29% ⫾ 1.5%) than in the two control groups (Group C: 3% ⫾ 0.2% and Group D: 7% ⫾ 0.4%), but there was no significant difference for mice receiving flutamide plus RPMI (Group A) vs flu-
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FIG. 2. Splenocyte secretion of IL2 and IFN-␥ in mice that were treated with or without flutamide and then challenged with irradiated B16 tumor vaccine or media alone. Splenocyte secretion of IFN-␥ was significantly higher in mice treated with flutamide plus irradiated B16 cells (Group B) than in the other three groups (P ⫽ 0.0024). Splenocyte secretion of IL2 also was highest in Group B but was not significantly different among the four groups (P ⫽ 0.2047).
tamide plus B16 (Group B). Although splenocyte proliferation was higher in mice receiving B16 alone (Group D) than in mice receiving placebo plus RPMI (Group C), this difference was not significant. Cytokine Secretion Assay Splenocyte secretion of IFN-␥ was significantly higher in mice treated with flutamide plus B16 cells (Group B) than in the other three groups (P ⫽ 0.0024) (Fig. 2). Splenocyte secretion of IL2 was also highest in Group B but did not differ significantly among the four groups (P ⫽ 0.2047) (Fig. 2). ADCC ADCC against B16 cells was significantly different (P ⫽ 0.0001) among the four groups. At a 5:1 effectortarget ratio, ADCC was 26% ⫾ 1.3% in mice receiving flutamide plus irradiated B16 (Group B), compared with 22% ⫾ 1.1% in mice receiving vehicle plus B16 (Group D); this difference approached statistical significance (P ⫽ 0.0746). The ADCC of Group B was significantly higher (P ⬍ 0.05) than ADCC values for Groups A (15% ⫾ 0.8%) and C (8% ⫾ 0.4%).
Tumor Growth Prevention Study At day 65, the survival rate was highest for the group of mice treated with irradiated B16 plus flutamide (Fig. 3). When the four mice left in this group were euthanized on day 65, there was no evidence of tumor on necropsy. In contrast, two of the three surviving mice in the irradiated B16 alone group were tumor-free on necropsy, and none of the surviving mice in the other two control groups were tumor-free on necropsy. DISCUSSION
In this murine study, androgen blockade enhanced immune responses (splenocyte proliferation, splenocytestimulated secretion of IFN-␥, and ADCC) to subsequent administration of an irradiated whole-cell melanoma vaccine. Other preclinical studies have examined the interaction between androgen and the immune system. In a murine study, androgen administration caused atrophy of the thymus and depression of lymphopoiesis [12], whereas castration increased the weight of the thymus and spleen [13]. Castrated male mice reportedly have a significant increase in splenic B lymphocytes compared with sham-operated male mice
HSUEH ET AL.: ANDROGEN BLOCKADE TO ENHANCE MELANOMA VACCINE
FIG. 3. Tumor growth prevention study. Mice were randomly assigned to four groups (n ⫽ 10 in each group). Each group underwent a 7-day treatment regimen that was followed by injection of vaccine and 7 days later by injection of live nonirradiated B16 cells. One group received 50 mg/kg/day flutamide and irradiated B16 tu). The second group received 50 mor vaccine (thick black line mg/kg/day flutamide and RPMI (vaccine control) (thin black line O). The third group received vehicle (1,2-propane-diol) and irradiated B16 tumor vaccine (thick gray line ). The fourth group received vehicle (1,2-propane-diol) and RPMI (thin gray line ).
[14]. Similarly, increased B-lymphocyte lymphopoiesis has been demonstrated in the bone marrow of castrated male mice [15]. A study using androgeninsensitive mice found a significant increase of B-cell precursors (CD45R⫹, IgM⫺) and mature B cells (sIgM⫹) in the bone marrow and a significant increase of mature B cells in the spleen [16]. Androgen also inhibited in vitro B-cell precursor expansion in the presence of stromal cells. Other murine studies have reported a reduction of bone marrow B-lymphocyte formation after androgen administration [17], and an increase in the release of IL2 and IFN-␥ by peripheral T cells in response to mitogen (Con A) after castration [13]. In the clinical setting, the effects of androgen on the immune system have been evaluated in patients undergoing androgen replacement therapy. In patients with Klinefelter’s syndrome, which is characterized by gynecomastia and underandrogenization [19], a decrease in the number of CD4⫹ cells and the CD4⫹/ CD8⫹ ratio as well as an increase in the number of CD8⫹ cells was observed following androgen replacement therapy [20, 21]. The decrease in CD4⫹/CD8⫹ ratio was also observed in rheumatoid arthritis patients receiving androgen replacement therapy [22]. Interestingly, total lymphocyte counts, CD4⫹ T cell number, and CD4⫹/CD8⫹ ratio were higher in patients with Klinefelter’s syndrome than in agematched healthy controls [20]. The effects of androgen on lymphocytes may be mediated by other immunoreactive signals. Expression of the androgen receptor (AR) could not be demonstrated in T lymphocytes, peripheral B cells, or in bone marrow
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B-cell precursors [23, 24]. We were unable to detect AR expression by immunohistochemical staining of 20 human melanoma biopsy specimens or by flow cytometry on B16 murine melanoma cells (E. Hsueh, unpublished data). However, other investigators have demonstrated AR expression in thymocytes [25]. Furthermore, action of androgen on lymphocytes may be mediated by the immune reactive signals from epithelial cells or macrophages [26, 27]. The results of our murine study are consistent with the reported effects of androgen blockade. The results obtained in the splenocyte proliferation assay indicated a generalized immunologic enhancement with androgen blockade, whereas the results obtained with IFN-␥ secretion assay suggested a specific immune enhancement to the immunizing agent (irradiated B16 tumor vaccine) after androgen blockade. A flutamideinduced specific immune enhancement to the immunizing agent was also observed in the ADCC experiment. The ability of androgen blockade to enhance host response to tumor vaccination is novel and, to our knowledge, has not been tested before. Our tumor growth prevention study demonstrated that androgen blockade with flutamide had a mild-tomoderate protective effect. This is not surprising because B16-F1 is a poorly immunogenic tumor whose growth may not be obliterated solely by immune manipulations. However, even a delay in growth is significant. In our study, all mice were male. The level of androgen in female mice might not have been high enough to yield a similar response to blockade with flutamide, and further evaluation is necessary to determine the effects of androgen blockade in females receiving immunotherapy. Nonetheless, the implications of the current study are potentially significant for males with cancer. The clinical combination of androgen blockade with immunotherapy might be especially important for patients with metastatic prostate cancer, in whom androgen blockade also has significant therapeutic role. In conclusion, androgen blockade can enhance immune responsiveness to tumor vaccine immunotherapy in this murine melanoma model. Our findings suggest that the clinical application of this combination might have an additive and synergistic effect on tumor killing. This attractive possibility merits clinical investigation of androgen blockade as adjuvant in trials of immunotherapy for advanced cancer. REFERENCES 1.
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