PURPOSE Despite significant strides in the identification and characterization of potential

PURPOSE Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma (MB), the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. tumors. However, murine Group 3 tumors had higher percentages of CD8+ PD-1+ T cells within the CD3 populace. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial Group 3 tumors compared to SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor CP-690550 efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1+ peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a designated increase in CD3+ T cells within the tumor microenvironment. CONCLUSIONS This is usually the first immunologic characterization of preclinical models of molecular subtypes of MB and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically given antibodies penetrate the brain tumor microenvironment. INTRODUCTION Medulloblastoma (MB), the most common malignant primary CP-690550 brain tumor of childhood, remains incurable in approximately one-third of patients despite surgical resection, radiation therapy and aggressive chemotherapy (1-3). Patients endure significant morbidities from such treatments, thus necessitating more targeted strategies that utilize accurate molecular subtype classification (1-3). Mediating consistent and safe treatment for MB patients represents the next goal in achieving an unmet need for the successful eradication of these malignancies (4). Immunotherapy presents an effective approach that has shown considerable advances in generating sustained and strong antitumor responses in malignant gliomas (5). However, the search of immune based strategies in pediatric brain tumors has been limited and to date, with few successful applications reported for MB patients (6, 7). A considerable obstacle in the development of MB immunotherapy has been a lack of understanding regarding the complex immunologic interactions that occur within the tumor microenvironment. Recent immunohistochemistry (IHC) and gene manifestation evidence has shed some light on the immunologic phenotype across MB subtypes and shown that tumor-associated macrophage and inflammatory gene upregulation could additionally be used to stratify the different molecular subgroups (8). These observations demonstrate that MB tumor subtypes contain highly distinct immune information, and further suggest that each subgroup may have different mechanisms of facilitating immune suppression and evasion. Despite a plethora of genetic and histopathological information from patient samples, an CP-690550 absence of relevant preclinical animal models has also hindered the investigation of promising immunotherapeutic targeting strategies of MB homolog 1 (gene, causing a loss of PTCH1 protein manifestation and constitutive SHH pathway activation (10). While homozygous mutations in the gene are embryonically lethal, heterozygotes (and a dominating unfavorable form of (13). Infected cells were implanted into the cerebella of immune deficient NOD scid IL-2 receptor gamma knockout (NSG) mice and formed tumors within 6-12 weeks (13). We adapted the aforementioned models for immunotherapeutic assessment through orthotopic transplantation of each tumor type into the cerebella of immune qualified C57BL/6 hosts. After several passages, we produced a large stock of Ptch1 MB and NSC MB tumor cells that could be stereotactically implanted to generate large cohorts of uniformly tumor-bearing animals. After successful adaptation and validation of these models, we characterized the tumor infiltrating immune cells in both animal models of MB. We analyzed both myeloid and T cell populations and further investigated markers of activated and suppressive immune cell phenotypes. Ptch1 MB tumors contained significantly increased frequencies of infiltrating dendritic cells, T cells and myeloid cells in comparison to NSC MB tumors. However, higher percentages CD8+ PD-1+ T cells of infiltrating CD3+ cells were identified in NSC IL10RB MB tumors. We show that blockade of the PD-1 conveying lymphocyte populace showed a significant antitumor benefit in intracranial NSC MB-bearing animals, but not in Ptch1 MB animals. Further analysis of treated tumors in both subtypes revealed anti-PD-1 monoclonal antibody to be only bound to PD-1+ T cells in peripheral lymphoid organs and absent on tumor infiltrating lymphocytes in the brain. PD-1 blockade also resulted in significant increases in infiltrating PD-1 unfavorable T cells within the tumor microenvironment. Our findings suggest that MB subgroups have distinct immune information that may require different immunotherapeutic targeting strategies to mediate antitumor immunity. MATERIALS AND METHODS Tumor Cells mutant mice and NSC MB (MP) cells were provided in collaboration with Dr. Robert Wechsler-Reya (Sanford-Burnham Research Institute, La Jolla, CA). The NSC MB.

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