[Clinicopathological characteristics of the CD8+ T lymphocytes infiltration and its mechanism in distinct molecular subtype of medulloblastoma].

OBJECTIVE: To investigate the characteristics of the CD8+ T cells infiltration from the 4 subtypes in medulloblastoma (MB), to analyze the relationship between CD8+ T cells infiltration and prognosis, to study the function of C-X-C motif chemokine ligand 11 (CXCL11) and its receptor in CD8+ T cells infiltration into tumors and to explore the potential mechanism, and to provide the necessary clinicopathological basis for exploring the immunotherapy of MB. METHODS: In the study, 48 clinical MB samples (12 cases in each of 4 subtypes) were selected from the multiple medical center from 2012 to 2019. The transcriptomics analysis for the tumor of 48 clinical samples was conducted on the NanoString PanCancer IO360TM Panel (NanoString Technologies). Immunohistochemistry (IHC) staining of formalin-fixed, paraffin-embedded sections from MB was carried out using CD8 primary antibody to analyze diffe-rential quantities of CD8+ T cells in the MB four subtypes. Through bioinformatics analysis, the relationship between CD8+T cells infiltration and prognosis of the patients and the expression differences of various chemokines in the different subtypes of MB were investigated. The expression of CXCR3 receptor on the surface of CD8+T cells in MB was verified by double immunofluorescence staining, and the underlying molecular mechanism of CD8+T cells infiltration into the tumor was explored. RESULTS: The characteristic index of CD8+T cells in the WNT subtype of MB was relatively high, suggesting that the number of CD8+T cells in the WNT subtype was significantly higher than that in the other three subtypes, which was confirmed by CD8 immunohistochemical staining and Gene Expression Omnibus (GEO) database analysis by using R2 online data analysis platform. And the increase of CD8+T cells infiltration was positively correlated with the patient survival. The expression level of CXCL11 in the WNT subtype MB was significantly higher than that of the other three subtypes. Immunofluorescence staining showed the presence of CXCL11 receptor, CXCR3, on the surface of CD8+T cells, suggesting that the CD8+T cells might be attracted to the MB microenvironment by CXCL11 through CXCR3. CONCLUSION: The CD8+T cells infiltrate more in the WNT subtype MB than other subtypes. The mechanism may be related to the activation of CXCL11-CXCR3 chemokine system, and the patients with more infiltration of CD8+T cells in tumor have better prognosis. This finding may provide the necessary clinicopathological basis for the regulatory mechanism of CD8+T cells infiltration in MB, and give a new potential therapeutic target for the future immunotherapy of MB.

GD2-Targeting CAR T-cell Therapy for Patients with GD2+ Medulloblastoma.

PURPOSE: Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. EXPERIMENTAL DESIGN: GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. RESULTS: GD2 was expressed in 82.68% of MB tumors. The SHH and G3-G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood-brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. CONCLUSIONS: Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

Molecular profile reveals immune-associated markers of medulloblastoma for different subtypes.

Medulloblastoma, a common pediatric malignant tumor, has been recognized to have four molecular subgroups [wingless (WNT), sonic hedgehog (SHH), group 3, group 4], which are defined by the characteristic gene transcriptomic and DNA methylomic profiles, and has distinct clinical features within each subgroup. The tumor immune microenvironment is integral in tumor initiation and progression and might be associated with therapeutic responses. However, to date, the immune infiltrative landscape of medulloblastoma has not yet been elucidated. Thus, we proposed MethylCIBERSORT to estimate the degree of immune cell infiltration and weighted correlation network analysis (WGCNA) to find modules of highly correlated genes. Synthesizing the hub genes in the protein-protein interaction (PPI) network and modules of the co-expression network, we identify three candidate biomarkers [GRB2-associated-binding protein 1 (GAB1), Abelson 1 (ABL1), and CXC motif chemokine receptor type 4 (CXCR4)] via the molecular profiles of medulloblastoma. Given this, we investigated the correlation between these three immune hub genes and immune checkpoint blockade response and the potential of drug prediction further. In addition, this study demonstrated a higher presence of endothelial cells and infiltrating immune cells in Group 3 tumor bulk. The above results will be conducive to better comprehending the immune-related pathogenesis and treatment of medulloblastoma.

Downregulation of ARID1B, a tumor suppressor in the WNT subgroup medulloblastoma, activates multiple oncogenic signaling pathways.

Medulloblastoma, a common pediatric malignant brain tumor, consists of four distinct molecular subgroups WNT, SHH, Group 3 and Group 4. Exome sequencing of 11 WNT subgroup medulloblastomas from an Indian cohort identified mutations in several chromatin modifier genes, including genes of the mammalian SWI/SNF complex. The genome of WNT subgroup tumors is known to be stable except for monosomy 6. Two tumors, having monosomy 6, carried a loss of function mutation in the ARID1B gene located on chromosome 6. ARID1B expression is also lower in the WNT subgroup tumors compared to other subgroups and normal cerebellar tissues that could result in haploinsufficiency. The short hairpin RNA-mediated knockdown of ARID1B expression resulted in a significant increase in the malignant potential of medulloblastoma cells. Transcriptome sequencing identified upregulation of several genes encoding cell adhesion proteins, matrix metalloproteases indicating the epithelial-mesenchymal transition. The ARID1B knockdown also upregulated ERK1/ERK2 and PI3K/AKT signaling with a decrease in the expression of several negative regulators of these pathways. The expression of negative regulators of the WNT signaling like TLE1, MDFI, GPX3, ALX4, DLC1, MEST decreased upon ARID1B knockdown resulting in the activation of the canonical WNT signaling pathway. Synthetic lethality has been reported between SWI/SNF complex mutations and EZH2 inhibition, suggesting EZH2 inhibition as a possible therapeutic modality for WNT subgroup medulloblastomas. Thus, the identification of ARID1B as a tumor suppressor and its downregulation resulting in the activation of multiple signaling pathways opens up opportunities for novel therapeutic modalities for the treatment of WNT subgroup medulloblastoma.

Changes in Peripheral Blood Regulatory T Cells and IL-6 and IL-10 Levels Predict Response of Pediatric Medulloblastoma and Germ Cell Tumors With Residual or Disseminated Disease to Craniospinal Irradiation.

PURPOSE: Radiation therapy (RT) modulates immune cells and cytokines, resulting in both clinically beneficial and detrimental effects. The changes in peripheral blood T lymphocyte subsets and cytokines during RT for pediatric brain tumors and the association of these changes with therapeutic outcomes have not been well described. METHODS AND MATERIALS: The study population consisted of children (n = 83, aged 3~18) with primary brain tumors (medulloblastoma, glioma, germ cell tumors (GCT), and central nervous system embryonal tumor-not otherwise specified), with or without residual or disseminated (R/D) diseases who were starting standard postoperative focal or craniospinal irradiation (CSI). Peripheral blood T lymphocyte subsets collected before and 4 weeks after RT were enumerated by flow cytometry. Plasma levels of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-α, interferon-γ, and IL-17A were measured by cytometric bead array. RESULTS: Patients with R/D lesions receiving CSI (n = 32) had a post-RT increase in the frequency of CD3+T and CD8+T cells, a decrease in CD4+T cells, and an increase in regulatory T cells (Tregs) and CD8+CD28- suppressor cells, which was more predominantly seen in these patients than in other groups. In the CSI group with such R/D lesions, consisting of patients with medulloblastoma and germ cell tumors, 19 experienced a complete response (CR) and 13 experienced a partial response (PR) on imaging at 4 weeks after RT. The post/pre-RT ratio of Tregs (P = .0493), IL-6 (P = .0111), and IL-10 (P = .0070) was lower in the CR group than in the PR group. Multivariate analysis revealed that the post/pre-RT ratios of Treg, IL-6, and IL-10 were independent predictors of CR (P < .0001, P = .018, P < .0001, respectively). The areas under the receiver operating curves and confidence intervals were 0.7652 (0.5831-0.8964), 0.7794 (0.5980-0.9067), and 0.7085 (0.5223-0.8552) for IL-6, IL-10, and Treg, respectively. The sensitivities of IL-6, IL-10, and Treg to predict radiotherapeutic responses were 100%, 92.3%, and 61.5%, and specificity was 52.6%, 57.9%, and 84.2%, respectively. CONCLUSIONS: CSI treatment to those with R/D lesions predominantly exerted an effect on antitumor immune response compared with both R/D lesion-free but exposed to focal or CSI RT and with R/D lesions and exposed to focal RT. Such CSI with R/D lesions group experiencing CR is more likely to have a decrease in immunoinhibitory molecules and cells than patients who only achieve PR. Measuring peripheral blood Treg, IL-6, and IL-10 levels could be valuable for predicting radiotherapeutic responses of pediatric brain tumors with R/D lesions to CSI for medulloblastoma and intracranial germ cell tumors.

Macrophages in SHH subgroup medulloblastoma display dynamic heterogeneity that varies with treatment modality.

Tumor-associated macrophages (TAMs) play an important role in tumor immunity and comprise of subsets that have distinct phenotype, function, and ontology. Transcriptomic analyses of human medulloblastoma, the most common malignant pediatric brain cancer, showed that medulloblastomas (MBs) with activated sonic hedgehog signaling (SHH-MB) have significantly more TAMs than other MB subtypes. Therefore, we examined MB-associated TAMs by single-cell RNA sequencing of autochthonous murine SHH-MB at steady state and under two distinct treatment modalities: molecular-targeted inhibitor and radiation. Our analyses reveal significant TAM heterogeneity, identify markers of ontologically distinct TAM subsets, and show the impact of brain microenvironment on the differentiation of tumor-infiltrating monocytes. TAM composition undergoes dramatic changes with treatment and differs significantly between molecular-targeted and radiation therapy. We identify an immunosuppressive monocyte-derived TAM subset that emerges with radiation therapy and demonstrate its role in regulating T cell and neutrophil infiltration in MB.

Development of a prognostic model based on an immunogenomic landscape analysis of medulloblastoma.

Medulloblastoma (MB) is one of the most common central nervous system tumors in children. At present, the vital role of immune abnormalities has been proved in tumorigenesis and progression. However, the immune mechanism in MB is still poorly understood. In the present study, 51 differentially expressed immune-related genes (DE-IRGs) and 226 survival associated immune-related genes (Sur-IRGs) were screened by an integrated analysis of multi-array. Moreover, the potential pathways were enriched by functional analysis, such as 'cytokine-cytokine receptor interaction', 'Ras signaling pathway', 'PI3K-Akt signaling pathway' and 'pathways in cancer'. Furthermore, 10 core IRGs were identified from DE-IRGs and Sur-IRGs. And the potential regulatory mechanisms of core IRGs were also explored. Additionally, a new prognostic model, including 7 genes (HDGF, CSK, PNOC, S100A13, RORB, FPR1, and ICAM2) based on IRGs, was established by multivariable COX analysis. In summary, our study revealed the underlying immune mechanism of MB. Moreover, we developed a prognostic model associated with clinical characteristics and could reflect the infiltration of immune cells.

Histone demethylase UTX/KDM6A enhances tumor immune cell recruitment, promotes differentiation and suppresses medulloblastoma.

The deregulation of epigenetic pathways has been implicated as a critical step in tumorigenesis including in childhood brain tumor medulloblastoma. The H3K27me3 demethylase UTX/KDM6A plays important roles in development and is frequently mutated in various types of cancer. However, how UTX regulates tumor development remains largely unclear. Here, we report the generation of a UTX-deleted mouse model of SHH medulloblastoma that demonstrates the tumor suppressor functions of UTX, which could be antagonized by the deletion of another H3K27me3 demethylase JMJD3/KDM6B. Intriguingly, UTX deletion in cancerous cerebellar granule neuron precursors (CGNPs) resulted in the impaired recruitment of host CD8+ T cells to the tumor microenvironment through a non-cell autonomous mechanism. In both mouse medulloblastoma models and in human medulloblastoma cells, we showed that UTX activates Th1-type chemokines, which are responsible for T cell migration. Surprisingly, our results showed that the depletion of cytotoxic CD8+ T cells did not affect mouse medulloblastoma growth. Nevertheless, the UTX/chemokine/T cell recruitment pathway we identified may be applied to many other cancers and may be important for improving cancer immunotherapy. In addition, UTX is required for the expression of NeuroD2 in precancerous progenitors, which encodes a potent proneural transcription factor. Overexpression of NEUROD2 in CGNPs decreased cell proliferation and increased neuron differentiation. We showed that UTX deletion led to impaired neural differentiation, which could coordinate with active SHH signaling to accelerate medulloblastoma development. Thus, UTX regulates both cell-intrinsic oncogenic processes and the tumor microenvironment in medulloblastoma. Our study provides insights into both medulloblastoma development and context dependent functions of UTX in tumorigenesis.

Design and Rationale for First-in-Human Phase 1 Immunovirotherapy Clinical Trial of Oncolytic HSV G207 to Treat Malignant Pediatric Cerebellar Brain Tumors.

Brain tumors represent the most common pediatric solid neoplasms and leading cause of childhood cancer-related morbidity and mortality. Although most adult brain tumors are supratentorial and arise in the cerebrum, the majority of pediatric brain tumors are infratentorial and arise in the posterior fossa, specifically the cerebellum. Outcomes from malignant cerebellar tumors are unacceptable despite aggressive treatments (surgery, radiation, and/or chemotherapy) that are harmful to the developing brain. Novel treatments/approaches such as oncolytic virotherapy are urgently needed. Preclinical and prior clinical studies suggest that genetically engineered oncolytic herpes simplex virus (HSV-1) G207 can safely target cerebellar malignancies and has potential to induce an antitumor immune response at local and distant sites of disease, including spinal metastases and leptomeningeal disease. Herein, we outline the rationale, design, and significance of a first-in-human immunotherapy Phase 1 clinical trial targeting recurrent cerebellar malignancies with HSV G207 combined with a single low-dose of radiation (5 Gy), designed to enhance virus replication and innate and adaptive immune responses. We discuss the unique challenges of inoculating virus through intratumoral catheters into cerebellar tumors. The trial utilizes a single arm open-label traditional 3 + 3 design with four dose cohorts. The primary objective is to assess safety and tolerability of G207 with radiation in recurrent/progressive malignant pediatric cerebellar tumors. After biopsy to prove recurrence/progression, one to four intratumoral catheters will be placed followed by a controlled-rate infusion of G207 for 6 h followed by the removal of catheters at the bedside. Radiation will be given within 24 h of virus inoculation. Patients will be monitored closely for toxicity and virus shedding. Efficacy will be assessed by measuring radiographic response, performance score, progression-free and overall survival, and quality of life. The data obtained will be invaluable in our efforts to produce more effective and less toxic therapies for children with high-grade brain tumors.

Cerebrospinal fluid cytokines in metastatic group 3 and 4 medulloblastoma.

BACKGROUND: Metastatic medulloblastoma (MB) portends a poor prognosis. Amongst the 4 molecular subtypes, Group 3 and Group 4 patients have a higher incidence of metastatic disease, especially involving the neuroaxis. At present, mechanisms underlying MB metastasis remain elusive. Separately, inflammation has been implicated as a key player in tumour development and metastasis. Cytokines and their inflammation-related partners have been demonstrated to act on autocrine and, or paracrine pathways within the tumour microenvironment for various cancers. In this study, the authors explore the involvement of cerebrospinal fluid (CSF) cytokines in Group 3 and 4 MB patients with disseminated disease. METHODS: This is an ethics approved, retrospective study of prospectively collected data based at a single institution. Patient clinicpathological data and corresponding bio-materials are collected after informed consent. All CSF samples are interrogated using a proteomic array. Resultant expression data of selected cytokines are correlated with each individual's clinical information. Statistical analysis is employed to determine the significance of the expression of CSF cytokines in Group 3 and 4 patients with metastatic MB versus non-metastatic MB. RESULTS: A total of 10 patients are recruited for this study. Median age of the cohort is 6.6 years old. Based on Nanostring gene expression analysis, 5 patients have Group 3 as their molecular subtype and the remaining 5 are Group 4. There are 2 non-metastatic versus 3 metastatic patients within each molecular subtype. Proteomic CSF analysis of all patients for both subtypes show higher expression of CCL2 in the metastatic group versus the non-metastatic group. Within the Group 3 subtype, the MYC-amplified Group 3 MB patients with existing and delayed metastases express higher levels of CXCL1, IL6 and IL8 in their CSF specimens at initial presentation. Furthermore, a longitudinal study of metastatic Group 3 MB observes that selected cytokines are differentially expressed in MYC-amplified metastatic Group 3 MB, in comparison to the non-MYC amplified metastatic Group 3 MB patient. CONCLUSION: This study demonstrates higher expression of selected CSF cytokines, in particular CCL2, in metastatic Group 3 and 4 MB patients. Although our results are preliminary, they establish a proof-of-concept basis for continued work in a larger cohort of patients affected by this devastating disease.

YBX1 Indirectly Targets Heterochromatin-Repressed Inflammatory Response-Related Apoptosis Genes through Regulating CBX5 mRNA.

Medulloblastomas arise from undifferentiated precursor cells in the cerebellum and account for about 20% of all solid brain tumors during childhood; standard therapies include radiation and chemotherapy, which oftentimes come with severe impairment of the cognitive development of the young patients. Here, we show that the posttranscriptional regulator Y-box binding protein 1 (YBX1), a DNA- and RNA-binding protein, acts as an oncogene in medulloblastomas by regulating cellular survival and apoptosis. We observed different cellular responses upon YBX1 knockdown in several medulloblastoma cell lines, with significantly altered transcription and subsequent apoptosis rates. Mechanistically, PAR-CLIP for YBX1 and integration with RNA-Seq data uncovered direct posttranscriptional control of the heterochromatin-associated gene CBX5; upon YBX1 knockdown and subsequent CBX5 mRNA instability, heterochromatin-regulated genes involved in inflammatory response, apoptosis and death receptor signaling were de-repressed. Thus, YBX1 acts as an oncogene in medulloblastoma through indirect transcriptional regulation of inflammatory genes regulating apoptosis and represents a promising novel therapeutic target in this tumor entity.

Tumor necrosis factor overcomes immune evasion in p53-mutant medulloblastoma.

Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-ß receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.

Locoregional delivery of CAR T cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma.

Recurrent medulloblastoma and ependymoma are universally lethal, with no approved targeted therapies and few candidates presently under clinical evaluation. Nearly all recurrent medulloblastomas and posterior fossa group A (PFA) ependymomas are located adjacent to and bathed by the cerebrospinal fluid, presenting an opportunity for locoregional therapy, bypassing the blood-brain barrier. We identify three cell-surface targets, EPHA2, HER2 and interleukin 13 receptor α2, expressed on medulloblastomas and ependymomas, but not expressed in the normal developing brain. We validate intrathecal delivery of EPHA2, HER2 and interleukin 13 receptor α2 chimeric antigen receptor T cells as an effective treatment for primary, metastatic and recurrent group 3 medulloblastoma and PFA ependymoma xenografts in mouse models. Finally, we demonstrate that administration of these chimeric antigen receptor T cells into the cerebrospinal fluid, alone or in combination with azacytidine, is a highly effective therapy for multiple metastatic mouse models of group 3 medulloblastoma and PFA ependymoma, thereby providing a rationale for clinical trials of these approaches in humans.

Worsening of anti-Hu paraneoplastic neurological syndrome related to anti-PD-1 treatment: Case report and review of literature.

We present an illustrative case of a 62-year-old woman with small cell lung cancer who developed progressive worsening of pre-existing anti-Hu antibody associated sensory neuronopathy after treatment with programmed cell death-1 (PD-1) inhibitor, nivolumab. We review the literature and identify 6 reported cases to understand the clinical outcomes of patients with anti-Hu paraneoplastic neurologic syndrome (PNS) treated with anti-PD-1 treatment. The PNS clinical spectrum comprised of encephalitis, a combination of sensory neuronopathy and anti-NMDAR encephalitis, isolated sensory neuronopathy, and encephalomyelitis. Immune checkpoint inhibitor have the potential to worsen pre-existing anti-Hu PNS and may promote the development of anti-Hu PNS.

Safety and efficacy of oncolytic HSV-1 G207 inoculated into the cerebellum of mice.

Primary malignant central nervous system (CNS) tumors are the leading cause of childhood cancer-related death and morbidity. While advances in surgery, radiation, and chemotherapy have improved the survival rates in children with malignant brain tumors, mortality persists in certain subpopulations and current therapies are associated with extreme morbidity. This is especially true for children with malignant infratentorial tumors. Accordingly, G207, a genetically engineered herpes simplex virus (HSV-1) capable of selectively targeting cancer cells has emerged as a promising therapeutic option for this patient population. Herein, we demonstrate that cerebellar inoculation of G207 was systemically non-toxic in an immunocompetent, HSV-1 sensitive mouse strain (CBA/J). Mice had neither abnormal brain/organ pathology nor evidence of G207 replication by immunohistochemistry at days 7 and 30 after cerebellar G207 inoculation. While a minute amount viral DNA was recovered in the cerebellum and brainstem of mice at day 7, no viral DNA persisted at day 30. Critically, G207 delivered to the cerebellum was able to target/treat the highly aggressive MYC-overexpressed group 3 murine medulloblastoma increasing survival vs controls. These results provide critical safety and efficacy data to support the translation of G207 for pediatric clinical trials in intractable cerebellar malignancies.

Massive clonal expansion of medulloblastoma-specific T cells during adoptive cellular therapy.

In both human and murine systems, we have developed an adoptive cellular therapy platform against medulloblastoma and glioblastoma that uses dendritic cells pulsed with a tumor RNA transcriptome to expand polyclonal tumor-reactive T cells against a plurality of antigens within heterogeneous brain tumors. We demonstrate that peripheral TCR Vß repertoire analysis after adoptive cellular therapy reveals that effective response to adoptive cellular therapy is concordant with massive in vivo expansion and persistence of tumor-specific T cell clones within the peripheral blood. In preclinical models of medulloblastoma and glioblastoma, and in a patient with relapsed medulloblastoma receiving adoptive cellular therapy, an early and massive expansion of tumor-reactive lymphocytes, coupled with prolonged persistence in the peripheral blood, is observed during effective therapeutic response to immunotherapy treatment.

Medulloblastoma rendered susceptible to NK-cell attack by TGFß neutralization.

BACKGROUND: Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor ß (TGF-ß). Here, we address this challenge in in vitro models of MB. METHODS: CB-derived NK cells were modified to express a dominant negative TGF-ß receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed. RESULTS: We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-ß-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-ß (decreased TGF-ß levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008). CONCLUSIONS: CB NK cells expressing a TGF-ß DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-ß-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.

Tumour-associated macrophages exhibit anti-tumoural properties in Sonic Hedgehog medulloblastoma.

Medulloblastoma, which is the most common malignant paediatric brain tumour, has a 70% survival rate, but standard treatments often lead to devastating life-long side effects and recurrence is fatal. One of the emerging strategies in the search for treatments is to determine the roles of tumour microenvironment cells in the growth and maintenance of tumours. The most attractive target is tumour-associated macrophages (TAMs), which are abundantly present in the Sonic Hedgehog (SHH) subgroup of medulloblastoma. Here, we report an unexpected beneficial role of TAMs in SHH medulloblastoma. In human patients, decreased macrophage number is correlated with significantly poorer outcome. We confirm macrophage anti-tumoural behaviour in both ex vivo and in vivo murine models of SHH medulloblastoma. Taken together, our findings suggest that macrophages play a positive role by impairing tumour growth in medulloblastoma, in contrast to the pro-tumoural role played by TAMs in glioblastoma, another common brain tumour.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

PURPOSE: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. METHODS: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. RESULTS: The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. CONCLUSIONS: Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.

An oncolytic measles virus-sensitive Group 3 medulloblastoma model in immune-competent mice.

Background: Oncolytic measles virus (MV) is effective in xenograft models of many tumor types in immune-compromised mice. However, no murine cell line exists that is tumorigenic, grows in immune-competent mice, and is killed by MV. The lack of such a model prevents an examination of the effect of the immune system on MV oncotherapy. Methods: Cerebellar stem cells from human CD46-transgenic immunocompetent mice were transduced to express Sendai virus C-protein, murine C-Myc, and Gfi1b proteins. The resultant cells were injected into the brain of NSG mice, and a cell line, called CSCG, was prepared from the resulting tumor. Results: CSCG cells are highly proliferative, and express stem cell markers. These cells are permissive for replication of MV and are killed by the virus in a dose- and time-dependent manner. CSCG cells form aggressive tumors that morphologically resemble medulloblastoma when injected into the brains of immune-competent mice. On the molecular level, CSCG tumors overexpress natriuretic peptide receptor 3 and gamma-aminobutyric acid type A receptor alpha 5, markers of Group 3 medulloblastoma. A single intratumoral injection of MV‒green fluorescent protein resulted in complete tumor regression and prolonged survival of animals compared with treatments with phosphate buffered saline (P = 0.0018) or heat-inactivated MV (P = 0.0027). Conclusions: This immune-competent model provides the first platform to test therapeutic regimens of oncolytic MV for Group 3 medulloblastoma in the presence of anti-measles immunity. The strategy presented here can be used to make MV-sensitive murine models of any human tumor for which the driving mutations are known.