Glioblastoma multiforme (GBM) is a highly malignant tumor with a poor outcome that is often positive for human cytomegalovirus (HCMV). GBM patients often have excessive numbers of neutrophils and macrophages near and within the tumor. Here, we characterized the cytokine patterns in the blood of GBM patients with and without Valganciclovir treatment. Furthermore, we determined whether neutrophil activation is related to HCMV status and patient outcome. Blood samples for analyses of cytokines and growth factors were collected from 42 GBM patients at the time of diagnosis (n = 42) and at weeks 12 and 24 after surgery. Blood neutrophils of 28 GBM patients were examined for CD11b expression. The levels of pro- and anti-inflammatory cytokines and chemokines-including interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-17A, transforming growth factor (TGF)-β1, interferon-γ, interferon-α, tumor necrosis factor α, and monocyte chemoattractant protein (MCP)-1were analyzed with a bead-based flow cytometry assay. During the first six months after surgery, neutrophil activity was increased in 12 patients and was unchanged or decreased in 16. Patients with increased neutrophil activity had enhanced IL-12p70, high grade HCMV and a shorter time to tumor progression (TTP) than patients without or decreased neutrophil activity (median TTP; 5.4 vs. 12 months, 95% confidence interval; 1.6-10 vs. 0.1-0.6, hazard ratio = 3 vs. 0.4, = 0.004). The levels of IL-12p70 were significantly decreased in Valganciclovir treated patients (n = 22, T 12W vs. T 24W, = 0.03). In conclusion, our findings suggest that neutrophil activation is an early sign of tumor progression in GBM patients.

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor. GBM is the most prevalent and the most aggressive primary malignancy of the central nervous system in adults. It is a highly vascularized and infiltrating tumor, rarely cured and prone to recurrence. The median duration of survival after diagnosis is less than 15 months, despite aggressive therapy consisting of surgical resection and concomitant radiotherapy and chemotherapy. 1 Surgical resection of GBMs is typically incomplete, as they are located in the brain and are highly infiltrative. Postoperative radiotherapy and chemotherapy fail to eradicate all remaining GBM cells. Thus, a breakthrough in identifying a new treatment option leading to a cure of this disease is still lacking.GBMs contain a subpopulation of highly tumorigenic cells with unlimited capacity for self-renewal that are commonly resistant to standard therapy. Phenotypically and functionally, these cells resemble neural stem cells and, when implanted in immunodeficient mice, can generate new tumors. As a result, they are referred to as glioma cancer initiating cells or glioma cancer stem cells (GCSCs) (reviewed in Lima et al. 2 ).Because of their apparent pivotal role in gliomagenesis and tumor recurrence after therapy, GCSCs are a major focus of research whose ultimate goal is to identify more effective therapies for GBM patients.GCSCs were first identified by their surface expression of CD133, based on the findings that these cells grow as neurospheres under nonadher...

$These authors share senior authorship.Keywords: cytomegalovirus, glioblastoma, immunosenescence, survival, T cells Abbreviations: GBM, glioblastoma multiforme; HCMV, human cytomegalovirus; PBMC, peripheral blood mononuclear cell; Valcyte, valganciclovir; WBC, white blood cell countPatients with glioblastoma multiforme (GBM) are immunosuppressed and have a broad range of immunological defects in both innate and adaptive immune responses. GBMs are frequently infected with human cytomegalovirus (HCMV), a virus capable of causing immunosuppression. In 42 HCMV-positive GBM patients in a clinical trial (VIGAS), we investigated T-cell phenotypes in the blood and assessed their relation to survival. Blood was collected before and 3, 12, and 24 weeks after surgery, and the frequency of T-cell subsets was compared with that in 26 age-matched healthy controls. GBM patients had lower levels of CD3 cells than the controls, but had significantly higher levels of CD4 C CD28 ¡ T cells before and 3 and 12 weeks after surgery and increased levels of CD4 C CD57 C and CD4 C CD57 C CD28 C T cells at all-time points. These T-cell subsets were associated with both immunosenescence and HCMV infection. GBM patients also had higher levels of gd T cells at all-times after surgery and lower levels of CD4 C CD25 C cells before and 3 weeks after surgery than healthy controls. Overall survival was significantly shorter in patients with higher levels of CD4 C CD28 ¡ T cells (p D 0.025), CD4 C CD57 C T (p D 0.025) cells, and CD4 C CD28 ¡ CD57 C CD28 ¡ T cells (p < 0.0004) at 3 weeks after surgery. Our findings indicate that signs of immunosenescence in the CD4 C compartment are associated with poor prognosis in patients with HCMV-positive GBMs and may reflect the HCMV activity in their tumors.