Immune-suppressive Myeloid Cells in the Glioma Microenvironment: Signaling Mechanisms and Novel Therapeutic Strategies

 描述（由申请人提供）：多形性胶质母细胞瘤（GBM）是成人中最常见的原发性恶性脑肿瘤;诊断后的中位生存期约为15-21个月。抗GBM免疫策略构成了新的和令人兴奋的治疗佐剂，以提高由于手术，化疗和放疗的生存率。然而，开发有效的抗GBM免疫应答以提高患者的存活率一直是一个挑战。由于可以诱导针对GBM抗原的全身免疫应答，因此认为临床失败是由于强大的GBM诱导的免疫抑制。GBM患者的免疫抑制由各种机制介导，包括在肿瘤微环境中积累的未成熟髓样细胞（IMC）。未成熟骨髓细胞的亚型是：（i）骨髓来源的抑制细胞（MDSC），（ii）免疫抑制性肿瘤相关巨噬细胞（TAM），和（iii）Tie 2+单核细胞（TEM）。GBM将未成熟的骨髓细胞募集到肿瘤微环境中，在那里它们抑制抗肿瘤免疫应答，例如，通过直接抑制T细胞效应子功能。另外的免疫抑制机制包括：TdR的积累，免疫抑制分子（即，吲哚胺2，3-双加氧酶1（IDO），细胞毒性T淋巴细胞抗原4（CTLA 4），和程序性死亡1受体配体（PDL 1），和细胞因子，（即，IL 10、TGFβ）。为了鉴定吸引免疫抑制性IMC进入GBM微环境的分泌因子，我们对内源性和可移植的小鼠和人GBM细胞进行了DNA微阵列分析，并鉴定了CXCL 12作为可能的候选物。我们还鉴定了GBM微环境内未成熟髓样细胞上的CXCR 4，即同源CXCL 12受体，支持CXCL 12/CXCR 4在将IMC吸引到GBM微环境中起重要作用的假设。为了确定CXCL 12-CXCR 4信号传导在GBM进展和调节抗GBM免疫疗法中所起的作用，我们提出使用免疫活性的、遗传工程化的内源性小鼠GBM模型。颅内肿瘤是由睡美人（SB）介导的插入人类GBM中发现的遗传改变诱导的。初步数据显示，来自可移植和SB诱导的GBM的条件培养基在体外引起高水平的IMC扩增。在体内GBM模型中，我们观察到GBM微环境和外周循环中IMC的积累。CXCR 4阻断显著延长了携带内源性GBM的小鼠的中位生存期。我们将使用CXCL 12和/或CXCR 4基因消融模型来检验以下假设：CXCL 12-CXCR 4信号传导轴在定性和定量确定GBM微环境的免疫特征方面起主要作用，因此对疾病进展具有深远影响。我们进一步假设阻断IMC的积累结合抗GBM免疫刺激策略将提供治疗恶性脑癌的强有力的辅助方法。

 DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults; median survival from diagnosis is ~15-21 months. Anti-GBM immune strategies constitute novel and exciting therapeutic adjuvants to improve survival due to surgery, chemo- and radiotherapy. However, it has been challenging to develop effective anti-GBM immune responses that translate into increased patients' survival. As systemic immune responses against GBM antigens can be induced, clinical failure is thought to be due to powerful GBM induced immune suppression. Immune suppression in GBM patients is mediated by various mechanisms that include immature myeloid cells (IMCs) that accumulate in the tumor microenvironment. Subtypes of immature myeloid cells are: (i) myeloid derived suppressor cells (MDSCs), (ii) immunosuppressive tumor associated macrophages (TAMs), and, (iii) Tie2+ monocytes (TEMs). GBMs recruit immature myeloid cells to the tumor microenvironment where they inhibit anti-tumor immune responses, for example, by directly inhibiting T-cell effector function. Additional immune suppressive mechanisms involve: accumulation of Tregs, immunosuppressive molecules (i.e., indoleamine2, 3-dioxygenase 1 (IDO), cytotoxic T-lymphocyte antigen 4 (CTLA4), and programmed death 1 receptor ligand (PDL1), and cytokines, (i.e., IL10, TGFβ). To identify secreted factors which attract immune-suppressive IMCs into the GBM microenvironment we performed DNA microarray analysis on endogenous and transplantable mouse and human GBM cells and identified CXCL12 as a possible candidate. We also identified CXCR4, the cognate CXCL12 receptor, on immature myeloid cells within the GBM microenvironment supporting the hypothesis that CXCL12/CXCR4 plays an important role in attracting IMCs to the GBM microenvironment. To ascertain the role played by CXCL12-CXCR4 signaling in GBM progression and in regulating anti-GBM immune therapies, we propose to use an immune competent, genetically engineered endogenous mouse GBM model. Intracranial tumors are induced by Sleeping Beauty (SB)-mediated insertion of genetic alterations found in human GBM. Preliminary data show that conditioned media from both transplantable and SB-induced GBM elicit a high level of IMCs' expansion in vitro. In GBM models in vivo, we observed accumulation of IMCs within the GBM microenvironment and in the peripheral circulation. CXCR4 blockade significantly prolonged median survival of mice bearing endogenous GBM. We will use CXCL12 and/or CXCR4 gene ablation models to test the hypothesis that CXCL12-CXCR4 signaling axis plays a major role in determining the immune profile, both qualitatively and quantitatively, of the GBM microenvironment and thus has profound effects on disease progression. We further hypothesize that blocking accumulation of IMCs in combination with anti-GBM immune stimulatory strategies will provide a powerful adjuvant approach to treat malignant brain cancer.