The full list of the 80 measured cytokines is shown in Table 1 and at http://www.raybiotech.com/human-cytokine-array-g5-4.html. follows for the representative cases shown: at pharmacologically relevant concentrations that are non-toxic to normal leukocytes. This is associated with a corresponding increase in mature dendritic cells. Interestingly, sulforaphane treatment had similar pro-inflammatory effects on normal monocytes in fresh media but specifically increased immature dendritic cells. Thus, we have used a simple model system to identify a novel contributor to glioblastoma immunosuppression for which a natural inhibitor exists that increases mature dendritic cell development at the expense of myeloid-derived suppressor cells when normal monocytes are exposed to glioma conditioned media. Introduction Glioblastoma (GBM) is a devastating disease with mean survival of 14 months despite optimal therapy.[1] Immunotherapies have emerged as promising therapeutic strategies for GBM.[2] Preclinical GBM immunotherapy studies have shown excellent results[3], but human clinical trials results have been more modest.[4] Local and systemic GBM-induced immunosuppression is a significant barrier to immunotherapy.[5] GDF5 Systemic immunosuppression in GBM patients reflects accumulations of immunosuppressive leukocytes such as myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs) which inhibit the proliferation and activation of T cells.[6, 7] MDSCs are derived from monocytes and include both monocytic and granulocytic variants.[8] In mice, they can be defined as CD11b+/Gr-1+/Ly6C+ (monocytic) or CD11b+/Gr-1+/Ly6G+ cells.[3] In humans, accepted MDSC surface marker profiles have evolved over the past decade. Monocytic MDSCs are currently best defined as CD11b+/CD14+/CD15-/HLA-DR- cells while granulocytic MDSCs are CD11b+/CD14-/CD15+ cells[9]. However, CD14+ cells almost universally also express CD11b and the necessity for excluding CD15+ cells from within monocytic MDSC definitions has only been widely accepted more recently. As result, many authors have relied only on CD14 and HLA-DR staining to identify monocytic MDSCs in cancer patients, either alone or as a surrogate after first identifying a population of MDSCs that are CD11b+/CD14+/CD15-/HLA-DR-. [10C12] Normal cells with similar surface marker phenotypes but without immunosuppressive function can occur. Therefore, true definition of MDSCs requires the demonstration of a functional ability to inhibit T cell proliferation in addition to surface marker profiling. MDSCs are present at low baseline levels in non-cancer patients with roles in preventing autoimmune states and moderating inflammatory reactions.[13, 14] Malignant tumors subvert this natural role of Menaquinone-7 MDSCs in order to protect themselves from tumor immunosurveillance.[15] Previously, we and others have shown that normal human monocytes co-cultured with GBM cells transform into both monocytic MDSCs (mMDSCs) and granulocytic MDSCs (gMDSCs).[16, 17] This same phenomenon is seen when syngeneic mouse monocytes are co-injected intracranially with GL261 murine glioma cells into C57BL/6 mice. The presence of increased monocytes in the mouse tumor environment from the time of implantation leads to increased tumor growth and increased intra-tumoral and systemic immunosuppressive MDSCs.[3] The mechanisms underlying MDSC accumulation in cancers such as GBM are not clear. GBM cells are known to secrete multiple immunomodulatory cytokines into the tumor microenvironment [16C19] though these are not generally increased in patients serum. Monocytes are continually trafficking in and out of the tumor microenvironment. [20] During this time they may undergo immunoeducation leading to their transformation into MDSCs.[3, 21] This process could occur secondary to cell-cell contact between na?ve monocytes and GBM cells or, alternatively, due to exposure to the cytokine-rich intra-tumoral environment.[3, 16] Findings in our glioma model suggest these MDSCs then re-enter the systemic circulation [3] where they inhibit T-cells proliferation and induce apoptosis in activated T-cells. Blocking the transformation of normal monocytes into MDSCs could have major implications in Menaquinone-7 immunotherapy. A non-immunosuppressed GBM or cancer patient may be able to mount a more robust anti-tumor response spontaneously or in response to a vaccine. However, studies of human MDSC biology in cancer have been hampered by both limited availability of patient material and cumbersome monocyte / cancer cell co-culture systems. Therefore, in this study we aimed to create a cell free human MDSC model and screen this model for targetable molecules contributing to MDSC development. Methods Glioblastoma cell culture Fresh human glioblastoma tumor tissue was obtained at surgery. Human specimens for this research were obtained with written, informed consent after approval of this project by the Mayo Clinic Institutional Review Board (Mayo Clinic IRB#12C003458). Single cell suspensions were generated by cutting tissue into small pieces followed by repeated aspiration through an 18-gauge needle. Cell cultures were originally established as brain tumor stem cell neurosphere lines in minimally hormonally supplemented serum-free media containing EGF and FGF as previously described.[22] They were subsequently transferred to DMEM with 1% penicillin/streptomycin and 10% fetal calf serum (FCS) Menaquinone-7 to generate.
The full list of the 80 measured cytokines is shown in Table 1 and at http://www