Purpose: The incidence of regulatory T cells (Treg) in intrinsic central nervous system malignancies is unknown. Immunotherapeutic approaches that inhibit the Treg population may be limited to a subset of patients with gliomas. Our hypothesis is that only the most malignant gliomas have a prominent glioma-infiltrating Treg population that contributes to the immunosuppressive biology and that the presence of Tregs is a negative prognostic variable. Experimental Design: We measured the incidence of Tregs in 135 glial tumors (including all pathologic types) in a glioma microarray using immunohistochemical analysis. Results were categorized according to the total number of Tregs within the tumors. Correlation of the presence of Tregs with prognosis was evaluated using univariate and multivariate analyses. Results: Tregs were not present in normal brain tissue and were very rarely found in low-grade gliomas and oligodendrogliomas. We observed significant differences in the prevalence of Tregs between astrocytic and oligodendroglial tumors, between tumors of different grades, and between different pathologic types of tumors. We identified Tregs most frequently in glioblastoma multiforme (GBM) but very rarely in low-grade astrocytomas. The presence of Tregs within GBMs did not alter the median survival in patients from whom the tumors were obtained. Conclusions: Treg infiltration differed significantly in the tumors according to lineage, pathology, and grade. Tregs seemed to have the highest predilection for tumors of the astrocytic lineage and specifically in the high-grade gliomas, such as GBM. In both univariate and multivariate analysis, the presence of Tregs in GBMs seemed to be prognostically neutral.

Purpose: The signal transducer and activator of transcription 3 (STAT3) is frequently overexpressed in most cancers, propagates tumorigenesis, and is a key regulator of immune suppression in cancer patients. We sought to determine the incidence of phosphorylated STAT3 (p-STAT3) expression in malignant gliomas of different pathologic types, whether p-STAT3 expression is a negative prognostic factor, and whether p-STAT3 expression influences the inflammatory response within gliomas. Methods: Using immunohistochemical analysis, we measured the incidence of p-STAT3 expression in 129 patients with gliomas of various pathologic types in a glioma tissue microarray. We categorized our results according to the total number of p-STAT3^expressing cells within the gliomas and correlated this number with the number of infiltrating T cells and T regulatory cells. We then evaluated the association between p-STAT3 expression and median survival time using univariate and multivariate analyses. Results: We did not detect p-STAT3 expression in normal brain tissues or low-grade astrocytomas. We observed significant differences in the incidence of p-STAT3 expression between the different grades of astrocytomas and different pathologic glioma types. p-STAT3 expression was associated with the population of tumor-infiltrating immune cells but not with that of T regulatory cells. On univariate analysis, we found that p-STAT3 expression within anaplastic astrocytomas was a negative prognostic factor. Conclusions: p-STAT3 expression is common within gliomas of both the astrocytic and oligodendroglial lineages and portends poor survival in patients with anaplastic astrocytomas. p-STAT3 expression differs significantly between gliomas of different pathologic types and grades and correlated with the degree of immune infiltration.A key transcription factor, signal transducer and activator of transcription (STAT3), has been shown to drive the fundamental components of tumorigenesis and metastasis by preventing apoptosis (by increasing survivin, BCL-XL, and MCL1 expression) and enhancing proliferation (by increasing c-Myc and cyclin D1/D2 expression; ref. 1), angiogenesis (by increasing vascular endothelial growth factor and hypoxia-inducible factor-1a expression), invasion (by increasing matrix metalloproteinase-2 and matrix metalloproteinase-9 expression), and metastasis (2, 3). Growth factors and cytokines, including interleukin (IL)-6, can activate Janus kinase 2, which subsequently activates STAT3 by phosphorylating the tyrosine residue in the STAT3 transactivation domain (4). Phosphorylated STAT3 (p-STAT3) then translocates into the nucleus and induces the expression of a variety of target genes. IL-6, which is expressed in the central nervous system (CNS) under a variety of conditions, such as hypoxia (5), traumatic and metabolic injury (6), and inflammation (7), has been shown to attract T cells to the CNS (8). Specifically, IL-6 signaling by means of STAT3 is tightly linked to the homing and migrational capacity of T cells (8)...

Purpose: Activation of signal transducers and activators of transcription 3 (STAT3) has been identified as a central mediator of melanoma growth and metastasis. We hypothesized that WP1066, a novel STAT3 blockade agent, has marked antitumor activity, even against the melanoma metastasis to brain, a site typically refractory to therapies. Experimental Design:The antitumor activities and related mechanisms of WP1066 were investigated both in vitro on melanoma cell lines and in vivo on mice with subcutaneously syngeneic melanoma or with intracerebral melanoma tumors. Results: WP1066 achieved an IC 50 of 1.6, 2.3, and 1.5 Amol/L against melanoma cell line A375, B16, and B16EGFRvIII, respectively.WP1066 suppressed the phosphorylation of Janus-activated kinase 2 and STAT3 (Tyr705) in these cells. Tumor growth in mice with subcutaneously established syngeneic melanoma was markedly inhibited by WP1066 compared with that in controls. Long-term survival (>78 days) was observed in 80% of mice with established intracerebral syngeneic melanoma treated with 40 mg/kg of WP1066 in contrast to control mice who survived for a median of 15 days. Although WP1066 did not induce immunologic memory or enhance humoral responses to EGFRvIII, this compound reduced the production of immunosuppressive cytokines and chemokines (transforming growth factor-h, RANTES, MCP-1, vascular endothelial growth factor), markedly inhibited natural and inducible Treg proliferation, and significantly increased cytotoxic immune responses of Tcells. Conclusions: The antitumor cytotoxic effects of WP1066 and its ability to induce antitumor immune responses suggest that this compound has potential for the effective treatment of melanoma metastatic to brain.Melanoma is a common and deadly tumor that despite the development of new therapeutic strategies, upon metastasis to the brain, is associated with a median survival time of <1 year. Furthermore, stage IV patients with brain metastasis are almost always excluded from participation in clinical trials. The Janus kinases (JAK)/signal transducers and activators of transcription (STAT)3 pathway is one of the key signaling pathways that drives the fundamental components of melanoma malignancy. The STAT3 protein is overexpressed in most cancers, including melanoma, and fosters tumorigenesis by preventing apoptosis (increases levels of the antiapoptotic proteins survivin, BCL-XL, and MCL1), enhancing proliferation (increases c-Myc and cyclin D1/D2 levels), angiogenesis [increases levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor], invasiveness (increases levels of matrix metalloproteinase 2 and matrix metalloproteinase 9), and metastasis (1, 2). Xie et al. (3) have shown that only highly metastatic melanoma cell lines, rather than poorly metastatic subtypes, overexpress matrix metalloproteinase 2, and have elevated levels of activated STAT3. Furthermore, blockade of activated STAT3 through expression of a dominant-negative STAT3 significantly suppresses invasiveness of the tu...

The activation of signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma malignancy, including immune suppression in melanoma patients. Increasing evidence also suggests that regulatory T cells (Tregs) are important in suppressing anti-tumor immunity and play a dominant role in negating efficacious immunotherapy approaches. We hypothesized that WP1066, a novel inhibitor of STAT3 signaling, reverses immune suppression through the inhibition of Tregs and that this contributes to the antitumor activity of these agents against melanoma brain metastases. We found that the mean percentage of peripheral blood mononuclear cells expressing phosphorylated STAT3 (p-STAT3) was significantly elevated in samples from patients with melanoma brain metastases compared to healthy donors, 16.13% ± 2.48% vs 4.17% ± 1.79%. The p-STAT3 inhibitor WP1066 enhanced CD3+ (which contained Tregs) but not CD8+ T cell cytotoxicity against human A375 melanoma cells, indicating that this p-STAT3 blockade agent did not directly activate CD8+ T cells. Furthermore, the p-STAT3 inhibitor did not enhance the cytotoxicity of CD3+CD25-T cells (from which Tregs were excluded), indicating that the enhanced cytotoxicity of WP1066 is secondary to its inhibition of Tregs. This was confirmed by demonstrating that WP1066 inhibited FoxP3+ Treg induction in a dose-dependent manner. Moreover, CD3+ T cells exhibited markedly enhanced levels of phosphorylated ZAP-70, a critical proximal signal in T cell activation, after exposure to WP1066. Similar effects were not observed in Treg-depleted CD3+CD25-T cell populations, confirming that the T cell activation by WP compounds is secondary to their inhibition of the Tregs. These results suggest that WP1066 enhances T cell cytotoxicity against melanoma through inhibition of Tregs.

Background: The signal transducer and activator of transcription 3 (STAT-3) is frequently overexpressed in cancer cells, propagates tumorigenesis, and is a key regulator of immune suppression in cancer patients. The presence of phosphorylated STAT-3 (p-STAT-3) in the tumor can induce p-STAT-3 in tumor-associated immune cells that can return to the circulatory system. We hypothesized that the number of peripheral blood mononuclear cells (PBMCs) displaying p-STAT-3 would be increased in glioma patients, which would correlate with the extent of tumorexpressed p-STAT-3, and that higher p-STAT-3 levels in peripheral blood would correlate with a higher fraction of immune-suppressive regulatory T cells (Tregs).