Therapy-Induced Changes in CXCR4 Expression in Tumor Xenografts Can Be Monitored Noninvasively with N-[11C]Methyl-AMD3465 PET.

PURPOSE: Chemokine CXCL12 and its receptor CXCR4 are constitutively overexpressed in human cancers. The CXCL12-CXCR4 signaling axis plays an important role in tumor progression and metastasis, but also in treatment-induced recruitment of CXCR4-expressing cytotoxic immune cells. Here, we aimed to demonstrate the feasibility of N-[11C]methyl-AMD3465 positron emission tomography (PET) to monitor changes in CXCR4 density in tumors after single-fraction local radiotherapy or in combination with immunization. PROCEDURE: TC-1 cells expressing human papillomavirus antigens E6 and E7 were inoculated into the C57BL/6 mice subcutaneously. Two weeks after tumor cell inoculation, mice were irradiated with a single-fraction 14-Gy dose of X-ray. One group of irradiated mice was immunized with an alpha-viral vector vaccine, SFVeE6,7, and another group received daily injections of the CXCR4 antagonist AMD3100 (3 mg/kg -intraperitoneal (i.p.)). Seven days after irradiation, all animals underwent N-[11C]methyl-AMD3465 PET. RESULTS: PET imaging showed N-[11C]methyl-AMD3465 uptake in the tumor of single-fraction irradiated mice was nearly 2.5-fold higher than in sham-irradiated tumors (1.07 ± 0.31 %ID/g vs. 0.42 ± 0.05 % ID/g, p < 0.01). The tumor uptake was further increased by 4-fold (1.73 ± 0.17 % ID/g vs 0.42 ± 0.05 % ID/g, p < 0.01) in mice treated with single-fraction radiotherapy in combination with SFVeE6,7 immunization. Administration of AMD3100 caused a 4.5-fold reduction in the tracer uptake in the tumor of irradiated animals (0.24 ± 0.1 % ID/g, p < 0.001), suggesting that tracer uptake is indeed due to CXCR4-mediated chemotaxis. CONCLUSION: This study demonstrates the feasibility of N-[11C]methyl-AMD3465 PET imaging to monitor treatment-induced changes in the density of CXCR4 receptors in tumors and justifies further evaluation of CXCR4 as a potential imaging biomarker for evaluation of anti-tumor therapies.

Noninvasive monitoring of cancer therapy induced activated T cells using [18F]FB-IL-2 PET imaging.

Cancer immunotherapy urgently calls for methods to monitor immune responses at the site of the cancer. Since activated T lymphocytes may serve as a hallmark for anticancer responses, we targeted these cells using the radiotracer N-(4-[18F]fluorobenzoyl)-interleukin-2 ([18F]FB-IL-2) for positron emission tomography (PET) imaging. Thus, we noninvasively monitored the effects of local tumor irradiation and/or immunization on tumor-infiltrating and systemic activated lymphocytes in tumor-bearing mice. A 10- and 27-fold higher [18F]FB-IL-2 uptake was observed in tumors of mice receiving tumor irradiation alone or in combination with immunization, respectively. This increased uptake was extended to several non-target tissues. Administration of the CXCR4 antagonist AMD3100 reduced tracer uptake by 2.8-fold, indicating a CXCR4-dependent infiltration of activated T lymphocytes upon cancer treatment. In conclusion, [18F]FB-IL-2 PET can serve as a clinical biomarker to monitor treatment-induced infiltration of activated T lymphocytes and, on that basis, may guide cancer immunotherapies.

Prediction model for regional or distant recurrence in endometrial cancer based on classical pathological and immunological parameters.

BACKGROUND: Adjuvant therapy increases disease-free survival in endometrial cancer (EC), but has no impact on overall survival and negatively influences the quality of life. We investigated the discriminatory power of classical and immunological predictors of recurrence in a cohort of EC patients and confirmed the findings in an independent validation cohort. METHODS: We reanalysed the data from 355 EC patients and tested our findings in an independent validation cohort of 72 patients with EC. Predictors were selected and Harrell's C-index for concordance was used to determine discriminatory power for disease-free survival in the total group and stratified for histological subtype. RESULTS: Predictors for recurrence were FIGO stage, lymphovascular space invasion and numbers of cytotoxic and memory T-cells. For high risk cancer, cytotoxic or memory T-cells predicted recurrence as well as a combination of FIGO stage and lymphovascular space invasion (C-index 0.67 and 0.71 vs 0.70). Recurrence was best predicted when FIGO stage, lymphovascular space invasion and numbers of cytotoxic cells were used in combination (C-index 0.82). Findings were confirmed in the validation cohort. CONCLUSIONS: In high-risk EC, clinicopathological or immunological variables can predict regional or distant recurrence with equal accuracy, but the use of these variables in combination is more powerful.

Antigen design enhances the immunogenicity of Semliki Forest virus-based therapeutic human papillomavirus vaccines.

Cellular immunity against cancer can be achieved with viral vector- and DNA-based immunizations. In preclinical studies, cancer vaccines are very potent, but in clinical trials these potencies are not achieved yet. Thus, a rational approach to improve cancer vaccines is warranted. We previously demonstrated that the relatively low intrinsic immunogenicity of DNA vaccines could be enhanced by inclusion of endoplasmic reticulum (ER) targeting and universal helper epitopes within the vaccine. We now evaluated whether an optimal antigen format, as defined in DNA vaccines, can further enhance the effectiveness of recombinant Semliki Forest virus (rSFV) vaccines. To this purpose, we generated, characterized and evaluated the efficacy of rSFV replicon particles expressing human papillomavirus E6 and/or E7 proteins fused to several helper T-cell epitopes and an ER targeting signal. Here, we show that inclusion of a helper cassette and an ER targeting signal enhanced protein stability and markedly augmented the frequencies of human papillomavirus-specific T cells. Even at an immunization dose of as low as 10(5) replicon particles, this novel vaccine achieved tumor regression and protection. Thus, even highly effective viral vector vaccines can benefit from an improved antigen format, based on the inclusion of defined helper epitopes and ER targeting.

Elevated serum CXCL16 is an independent predictor of poor survival in ovarian cancer and may reflect pro-metastatic ADAM protease activity.

BACKGROUND: In certain cancers, expression of CXCL16 and its receptor CXCR6 associate with lymphocyte infiltration, possibly aiding anti-tumour immune response. In other cancers, CXCL16 and CXCR6 associate with pro-metastatic activity. In the current study, we aimed to characterise the role of CXCL16, sCXCL16, and CXCR6 in ovarian cancer (OC). METHODS: CXCL16/CXCR6 expression was analysed on tissue microarray containing 306 OC patient samples. Pre-treatment serum sCXCL16 was determined in 118 patients using ELISA. In vitro, (primary) OC cells were treated with an ADAM-10/ADAM-17 inhibitor (TAPI-2) and an ADAM-10-specific inhibitor (GI254023x), whereupon CXCL16 levels were evaluated on the cell membrane (immunofluorescent analysis, western blots) and in culture supernatants (ELISA). In addition, cell migration was assessed using scratch assays. RESULTS: sCXCL16 independently predicted for poor survival (hazard ratio=2.28, 95% confidence interval=1.29-4.02, P=0.005), whereas neither CXCL16 nor CXCR6 expression correlated with survival. Further, CXCL16/CXCR6 expression and serum sCXCL16 levels did not associate with lymphocyte infiltration. In vitro inhibition of both ADAM-17 and ADAM-10, but especially the latter, decreased CXCL16 membrane shedding and strongly reduced cell migration of A2780 and cultured primary OC-derived malignant cells. CONCLUSIONS: High serum sCXCL16 is a prognostic marker for poor survival of OC patients, possibly reflecting ADAM-10 and ADAM-17 pro-metastatic activity. Therefore, serum sCXCL16 levels may be a pseudomarker that identifies patients with highly metastatic tumours.

Interleukin-6 receptor and its ligand interleukin-6 are opposite markers for survival and infiltration with mature myeloid cells in ovarian cancer.

An increased level of interleukin-6 (IL-6) in epithelial ovarian cancer (EOC) is correlated with a worse prognosis. IL-6 stimulates tumor-growth and inflammation. We investigated the intricate interaction between the IL-6 signaling pathway and tumor-infiltrating myeloid cells (TIMs) to determine their prognostic impact in EOC. 160 EOC samples were analyzed for the expression of IL-6, its receptor (IL-6R) and downstream signaling via pSTAT3 by immunohistochemistry. Triple color immunofluorescence confocal microscopy was used to identify myeloid cell populations by CD14, CD33, and CD163. The relationship between these markers, tumor-infiltrating immune cells, clinical-pathological characteristics and survival was investigated. EOC displayed a dense infiltration with myeloid cells, in particular of the CD163+ type. The distribution pattern of all myeloid subtypes was comparable among the different histological subtypes. Analysis of the tumor cells revealed a high expression of IL-6R in 15% and of IL-6 in 23% of patients. Interestingly, tumors expressing IL-6 or IL-6R formed two different groups. Tumors with a high expression of IL-6R displayed low mature myeloid cell infiltration and a longer disease-specific survival (DSS), especially in late stage tumors. High expression of IL-6R was an independent prognostic factor for survival by multivariate analyses (hazard ratio = 0.474, p = 0.011). In contrast, tumors with high epithelial IL-6 expression displayed a dense infiltration of mature myeloid cells and were correlated with a shorter DSS. Furthermore, in densely CD8+ T-cell infiltrated tumors, the ratio between these lymphoid cells and CD163+ myeloid cells was predictive for survival. Thus, IL-6 and IL-6R are opposite markers for myeloid cell infiltration and survival.

Cost-effectiveness of the prophylactic HPV vaccine: an application to the Netherlands taking non-cervical cancers and cross-protection into account.

Despite an effective screening programme, 600-700 women are still diagnosed with cervical cancer in the Netherlands each year. In 2009 a prophylactic vaccine against HPV-type 16 and 18 was implemented in the national immunisation programme to decrease the incidence of cervical cancer. There is evidence that infections with several oncogenic HPV types other than the vaccine types 16 and 18 are also prevented by vaccination, also known as cross-protection. Besides cervical cancer, HPV can also cause cancers at other sites such as the oropharynx, vulva, vagina and the anus/anal area. In this study we estimated the maximum health and economic benefits of vaccinating 12-year old girls against infection with HPV, taking cross-protection and non-cervical cancers into account. In the base-case, we found an incremental cost ratio (ICER) of €5815 per quality adjusted life year (QALY). Robustness of this result was examined in sensitivity analysis. The ICER proved to be most sensitive to vaccine price, discounting rates, costs of cervical cancer and to variation in the disutility of cervical cancer.

The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are often found in tumours, presumably reflecting an immune response against the tumour. We carried out a systematic review and meta-analysis, aiming to establish pooled estimates for survival outcomes based on the presence of TILs in cancer. METHODS: A Pubmed and Embase literature search was designed. Studies were included, in which the prognostic significance of intratumoural CD3+, CD4+, CD8+, and FoxP3+ lymphocytes, as well as ratios between these subsets, were determined in solid tumours. RESULTS: In pooled analysis, CD3+ TILs had a positive effect on survival with a hazard ratio (HR) of 0.58 (95% confidence interval (CI) 0.43-0.78) for death, as did CD8+ TILs with a HR of 0.71 (95% CI 0.62-0.82). FoxP3+ regulatory TILs were not linked to overall survival, with a HR of 1.19 (95% CI 0.84-1.67). The CD8/FoxP3 ratio produced a more impressive HR (risk of death: HR 0.48, 95% CI 0.34-0.68), but was used in relatively few studies. Sample size and follow-up time seemed to influence study outcomes. CONCLUSION: Any future studies should be carefully designed, to prevent overestimating the effect of TILs on prognosis. In this context, ratios between TIL subsets may be more informative.

Immunological and clinical effects of vaccines targeting p53-overexpressing malignancies.

Approximately 50% of human malignancies carry p53 mutations, which makes it a potential antigenic target for cancer immunotherapy. Adoptive transfer with p53-specific cytotoxic T-lymphocytes (CTL) and CD4(+) T-helper cells eradicates p53-overexpressing tumors in mice. Furthermore, p53 antibodies and p53-specific CTLs can be detected in cancer patients, indicating that p53 is immunogenic. Based on these results, clinical trials were initiated. In this paper, we review immunological and clinical responses observed in cancer patients vaccinated with p53 targeting vaccines. In most trials, p53-specific vaccine-induced immunological responses were observed. Unfortunately, no clinical responses with significant reduction of tumor-burden have occurred. We will elaborate on possible explanations for this lack of clinical effectiveness. In the second part of this paper, we summarize several immunopotentiating combination strategies suitable for clinical use. In our opinion, future p53-vaccine studies should focus on addition of these immunopotentiating regimens to achieve clinically effective therapeutic vaccination strategies for cancer patients.

Identification of genes and pathways associated with cytotoxic T lymphocyte infiltration of serous ovarian cancer.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are predictors of disease-specific survival (DSS) in ovarian cancer. It is largely unknown what factors contribute to lymphocyte recruitment. Our aim was to evaluate genes and pathways contributing to infiltration of cytotoxic T lymphocytes (CTLs) in advanced-stage serous ovarian cancer. METHODS: For this study global gene expression was compared between low TIL (n=25) and high TIL tumours (n=24). The differences in gene expression were evaluated using parametric T-testing. Selectively enriched biological pathways were identified with gene set enrichment analysis. Prognostic influence was validated in 157 late-stage serous ovarian cancer patients. Using immunohistochemistry, association of selected genes from identified pathways with CTL was validated. RESULTS: The presence of CTL was associated with 320 genes and 23 pathways (P<0.05). In addition, 54 genes and 8 pathways were also associated with DSS in our validation cohort. Immunohistochemical evaluation showed strong correlations between MHC class I and II membrane expression, parts of the antigen processing and presentation pathway, and CTL recruitment. CONCLUSION: Gene expression profiling and pathway analyses are valuable tools to obtain more understanding of tumour characteristics influencing lymphocyte recruitment in advanced-stage serous ovarian cancer. Identified genes and pathways need to be further investigated for suitability as therapeutic targets.

Cost-effectiveness of prophylactic vaccination against human papillomavirus 16/18 for the prevention of cervical cancer: adaptation of an existing cohort model to the situation in the Netherlands.

Cervical cancer is one of the most prevalent cancers among women worldwide. Implementation of an HPV-vaccination strategy targeting the major oncogenic types 16 and 18 that cause cervical cancer is generally expected to significantly reduce the burden of cervical cancer disease. Here we estimate the costs, savings and health gains with the addition of HPV-16/18 vaccination to the already existing Dutch screening programme. In the base-case analysis, it was estimated that implementation of an HPV-16/18 vaccine would result in an incremental cost-effectiveness ratio (ICER) of euro22,700 per life-year gained (LYG). In sensitivity analysis, the robustness of our finding of favourable cost-effectiveness was established. The ICER appeared sensitive to the vaccine price, discount rate and duration of vaccine-induced protection. From our results, it validly follows that immunization of 12-year-old Dutch girls against HPV-16/18 infection is a cost-effective strategy for protecting against cervical cancer.

Viral vector-based prime-boost immunization regimens: a possible involvement of T-cell competition.

Vaccination with recombinant viral vectors may be impeded by preexisting vector-specific immunity or by vector-specific immunity induced during the priming immunization. It is assumed that virus-neutralizing antibodies represent the principal effector mechanism of vector-specific immunity, while killing of infected cells by vector-specific cytotoxic T lymphocytes (CTLs) has also been suggested. Using recombinant Semliki Forest virus (rSFV) expressing E6E7 antigen from human papillomavirus, we demonstrate that secondary immune responses against E6E7 are neither affected by vector-specific antibodies nor by CTL-mediated killing of infected cells. Instead, the presence of the antigen during the prime immunization appeared to be the main determinant for the boosting efficacy. After priming with rSFVeE6,7, a homologous booster stimulated the primed E6E7-specific CTL response and induced long-lasting memory. Passively transferred SFV-neutralizing antibodies did not inhibit E6E7-specific CTL responses, although transgene expression was strongly reduced under these conditions. Conversely, in mice primed with irrelevant rSFV, induction of E6E7-specific CTLs was inhibited presumably due to vector-specific responses induced by the priming immunization. When during the priming with irrelevant rSFV, E7-protein was co-administered, the inhibitory effect of vector-specific immunity was abolished. These results suggest that, apart from vector-specific antibodies or killing of infected cells, T-cell competition may be involved in determining the efficacy of viral vector-based prime-boost immunization regimens.

Frequencies and role of regulatory T cells in patients with (pre)malignant cervical neoplasia.

Oncogenic human papillomavirus (HPV)-infection is crucial for developing cervical cancer and its precursor lesions [cervical intraepithelial neoplasia (CIN)]. Regulatory T cells (T(regs)) might be involved in the failure of the immune system to control the development of HPV-induced cancer. We investigated frequencies, phenotype and activity of T(regs) in patients with cervical neoplasia. CIN and cervical cancer patients showed increased CD4(+)/CD25(high) T cell frequencies in peripheral blood and CD4(+) T cell fraction. These CD4(+)/CD25(high) T cells represent T(regs) as demonstrated by their low proliferation rate, low interferon (IFN)-gamma/interleukin (IL)-10 ratio, high expression of CD45RO, GITR, CTLA-4, forkhead box P3 (FoxP3) and low CD45RA expression. Moreover, in HPV16(+) cervical cancer patients, in-vitro depletion of CD25(+) T cells resulted in increased IFN-gamma T cell responses against HPV16 E6- and E7 peptides. Thus, increased frequencies of T(regs) in cervical cancer patients may indeed suppress HPV-specific immunity. Longitudinal analysis of CD4(+)/CD25(high) T cell frequencies in patients showed a modest decline 1 year after curative surgery or chemoradiation. This study demonstrates increased frequencies and suppressive activity of T(regs) in cervical cancer. These results imply that T(regs) may suppress the immune control of cervical neoplasia and furthermore that suppression of immunity by T(regs) will be another hurdle to overcome in therapeutic immunization strategies against cervical neoplasia.

A comparative study on the immunotherapeutic efficacy of recombinant Semliki Forest virus and adenovirus vector systems in a murine model for cervical cancer.

Currently, various therapeutic strategies are being explored as a potential means to immunize against metastatic malignant cells or even primary tumours. Using recombinant viral vectors systems or protein-based immunization approaches, we are developing immunotherapeutic strategies against cervical cancer or premalignant cervical disease, as induced by high-risk type human papillomaviruses (HPVs). We previously demonstrated that immunization of mice with recombinant replication-defective Semliki Forest virus (rSFV) encoding a fusion protein of HPV16 E6 and -E7 (SFV-eE6,7) induces strong cytotoxic T-lymphocyte (CTL) activity and eradication of established HPV-transformed tumours. In this study, we compared the antitumour efficacy of SFV-eE6,7 with that of a recombinant adenovirus (rAd) type 5 vector, expressing the same antigen construct (Ad-eE6,7). Prime-boosting with SFV-eE6,7 resulted in higher precursor CTL frequencies and CTL activity compared to prime-boosting with Ad-eE6,7 and also in murine tumour treatment experiments SFV-eE6,7 was more effective than Ad-eE6,7. To elicit a therapeutic effect with Ad-eE6,7, 100/1000-fold higher doses were needed compared to SFV-eE6,7. In vivo T-cell depletion experiments demonstrated that these differences could not be explained by the induction of a different type of effector cells, since CD8+ T cells were the main effector cells involved in the protection against tumour growth in both rSFV- and rAd-immunized mice. Also comparable amounts of in vivo transgene expression were found upon immunization with rSFV and rAd encoding the reportor gene luciferase. However, anti-vector responses induced by a single injection with rAd resulted in a more than 3-log decrease in luciferase expression after a second injection of rAd. With rSFV, transgene expression was inhibited by only one to two orders of magnitude in preinjected mice. As an antigen-specific booster immunization strongly increases the level of the CTL response and is essential for efficient induction of immunological memory, it is likely that (part of) the difference in efficacy between rSFV and rAd type 5 can be ascribed to a diminished efficacy of the booster immunization in the case of rAd due to anti-vector antibody responses.

Immunologic aspect of ovarian cancer and p53 as tumor antigen.

Ovarian cancer represents the fifth leading cause of death from all cancers for women. During the last decades overall survival has improved due to the use of new chemotherapy schedules. Still, the majority of patients die of this disease. Research reveals that ovarian cancer patients exhibit significant immune responses against their tumor. In this review the knowledge obtained thus far on the interaction of ovarian cancer tumor cells and the immune system is discussed. Furthermore the role of p53 as tumor antigen and its potential role as target antigen in ovarian cancer is summarized. Based on the increased knowledge on the role of the immune system in ovarian cancer major improvements are to be expected of immunotherapy based treatment of this disease.

Induction of human papilloma virus E6/E7-specific cytotoxic T-lymphocyte activity in immune-tolerant, E6/E7-transgenic mice.

Despite promising preclinical results of various therapeutic anticancer immunization strategies, these approaches may not be effective enough to eradicate tumors in cancer patients. While most animal models are based on fast-growing transplantable tumors, malignancies in, for example, cervical cancer patients in general develop much more slowly, which may lead to immune suppression and/or immune tolerance. As a consequence, the immunomodulating signal of any therapeutic immunization regimen should be sufficiently potent to overcome this immunocompromised condition. In previous studies, we demonstrated that an experimental vaccine against human papillomavirus (HPV)-induced cervical cancer, based on Semliki Forest virus (SFV), induces robust HPV-specific cellular immune responses in mice. Now we studied whether this strategy is potent enough to also prime a cellular immune response in immune-tolerant HPV transgenic mice, in which CTL activity cannot be induced using protein or DNA vaccines. We demonstrate that, depending on the route of immunization, SFV-expressing HPV16 E6 and E7 indeed has the capacity to induce HPV16 E7-specific cytotoxic T cells in HPV-transgenic mice.

A potential role of macrophage activation in the treatment of cancer.

One of the functions of macrophages is to provide a defense mechanism against tumor cells. In the last decades the mechanism of tumor cell killing by macrophages have been studied extensively. The tumor cytotoxic function of macrophages requires stimulation either with bacterial cell wall products such as lipopolysaccharide (LPS) or muramyldipeptide (MDP) or with cytokines such as interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Activated macrophages secrete several substances that are directly involved in tumor cell killing i.e. tumor necrosis factor (TNF) and nitric oxide (NO). On the other hand, substances are secreted that are able to stimulate tumor cell growth, depending on the stage and the nature of the tumor. Several clinical trials have been performed aiming at the activation of macrophages or dendritic cells, a subpopulation of the macrophages. In this review we will summarize and discuss experimental studies and clinical trials based on the activation of macrophages.

Immunization strategy against cervical cancer involving an alphavirus vector expressing high levels of a stable fusion protein of human papillomavirus 16 E6 and E7.

We are developing immunization strategies against cervical carcinoma and premalignant disease, based on the use of recombinant Semliki Forest virus (SFV) encoding the oncoproteins E6 and E7 from high-risk human papilloma viruses (HPV). Thus far, protein-based, as well as genetic immunization studies have demonstrated low to moderate cellular immune responses against E6 and E7. To improve these responses, we modified the structure and expression level of the E6 and E7 proteins produced by the SFV vector. Specifically, a construct was generated encoding a fusion protein of E6 and E7, while furthermore a translational enhancer was included (enhE6,7). Infection of cells with recombinant SFV-enhE6,7 resulted in the production of large amounts of the E6,7 fusion protein. The fusion protein was more stable than either one of the separate proteins. Immunization of mice with SFV-enhE6,7 resulted in strong, long-lasting HPV-specific cytotoxic T lymphocyte responses. Tumor challenge experiments in mice demonstrated that immunization with SFV-enhE6,7 resulted in prevention of tumor outgrowth and subsequent protection against tumor re-challenge.

Replication-defective recombinant Semliki Forest virus encoding GM-CSF as a vector system for rapid and facile generation of autologous human tumor cell vaccines.

This paper describes the production of recombinant Semliki Forest virus encoding murine or human granulocyte-macrophage colony-stimulating factor (GM-CSF) and the capacity of these vectors to transduce murine and human tumor cells ex vivo. High-titer stocks (up to 3 x 10(9) particles/ml) of conditionally infective, replication-defective, recombinant SFV particles were generated using the SFV Helper-2 system. It is shown that the recombinant SFV/GM-CSF virus, as well as recombinant SFV carrying the beta-galactosidase reporter gene, efficiently transduce both murine tumor cell lines as well as primary human renal carcinoma cells. Using ELISA's specific for GM-CSF, levels of GM-CSF production by the cells were determined. Levels of murine GM-CSF (mGM-CSF) produced by SFV/mGM-CSF transduced renal cell cancer cultures were equal to or higher than corresponding levels reported in the literature after transduction of similar renal carcinoma cell cultures using a retroviral vector system. The biological activity of GM-CSF was demonstrated by using cells which are dependent on GM-CSF for growth and by using primary bone marrow cells. All the transduced cell cultures (including the human renal cell carcinoma samples) produced GM-CSF for up to at least 4 days after transduction. The results imply that the recombinant SFV system can be used for rapid and facile preparation of autologous cancer cell vaccines.

Expression of cyclooxygenase-2 and inducible nitric oxide synthase in human ovarian tumors and tumor-associated macrophages.

This study investigates whether and to what extent cyclooxygenase type-2 (COX-2) and inducible nitric oxide-synthase (iNOS), both known to have an immunosuppressive effect, are expressed in human ovarian tumors. Because COX-2 and iNOS can be expressed by activated macrophages, the presence of tumor-associated macrophages and the expression of COX-2 and iNOS by these tumor-associated macrophages were determined. The results obtained may provide insight into the function of COX-2 and iNOS expression by tumors. The expression of COX-2 and iNOS in tumor cells and macrophages was assessed in 18 malignant, 15 borderline, and 14 benign human ovarian tumors by immunohistochemical staining of frozen tissue sections. The intra- and peritumoral macrophages were stained using an anti-CD68 monoclonal antibody. Most of the malignant tumors (15 of 18), 10 of 15 borderline, and 9 of 14 benign tumors showed COX-2 expression in the epithelial cells, a result which indicates that COX-2 expression is not exclusive to malignancy. In addition, COX-2 staining was more intense in the epithelial cells of benign and borderline tumors than in malignant tumors. Weak iNOS staining was observed in 5 of 18 malignant, 4 of 15 borderline, and 5 of 14 benign tumors. The number of tumor-associated macrophages varied widely between the different tumors. The highest number of tumor-associated macrophages (> or =20/0.125 mm(2)) was observed in malignant tumors, whereas low to moderate intra- and peritumoral macrophage infiltration (5-20/0.125 mm(2)) was observed in the borderline and benign tumors. COX-2-positive tumor-associated macrophages were found in 3 of 18 malignant tumors, 7 of 15 borderline tumors, and 1 of 14 benign tumors. The number of COX-2-positive tumor-associated macrophages ranged from 3 to 30% of the total macrophage population. Some malignant (4 of 18), borderline (5 of 15), and benign (2 of 14) tumors contained iNOS-positive macrophages. Notable was that COX-2- and iNOS-positive macrophages were predominantly located in the tumor stroma, the regions between tumor and stroma, and in the lumina of the tumor when located in the tumor tissue. These data indicate that not only malignant but also borderline and benign ovarian tumors can exhibit increased levels of COX-2 and iNOS expression. In addition, a small proportion of the tumor-associated macrophages found in malignant, borderline, and benign tumors seems to be in an activated state, judged by their iNOS and COX-2 expression. This subpopulation of tumor-associated macrophages was invariably located in the tumor stroma or in the lumina of the tumor, specifically suggesting that macrophages outside the tumor can be tumor cytotoxic.