Bone marrow-derived myeloid cell dysregulation in malignant progression of glioma

项目摘要 低级别胶质瘤(LGG)是一组异质性的原发中枢神经系统肿瘤，是 青少年和青壮年(Aya)人群中最常见的实体肿瘤。LGG可以发展到高水平- 分级胶质瘤(HGG)通过一个称为恶性转化(MT)的过程，导致惨淡的预后。 LGG发生MT的机制尚不清楚。与LGG相比，我们和其他人已经展示了 HGG增加了骨髓来源的髓系细胞(BMDM)和髓系来源的水平 外周循环中的抑制细胞(MDSCs)，肿瘤内肿瘤相关物质的丰富 巨噬细胞(TAMs)，CD8+T细胞和自然杀伤(NK)细胞稀少。尽管已知髓系细胞 尽管这些细胞在胶质瘤进展过程中积聚，但目前尚不清楚这些细胞是否在驱动MT方面起到了因果作用。中环 假设：抗肿瘤反应性CD8+T和NK细胞依赖的免疫监视的减少是 在AYA患者中负责LGG向HGG的恶变，这些减少是由 肿瘤浸润性骨髓基质细胞的侵袭和免疫抑制活性增强。我们已经确认了两个 导致这些BMDM表型的独立的髓系相关通路，我们建议 通过以下两个目的验证中心假设：目的1.描绘BMDM细胞的依赖性 DNA结合蛋白2抑制物(ID2)的分化。使用Aya RCAS/TV-a胶质瘤小鼠模型，我们将 在MT之前和期间BMDM中的ID2基因被击倒。我们将检查对亲肿瘤的髓系细胞和 评价不同时间肿瘤内T细胞和NK细胞在血、脾和骨髓中的动员情况 ScRNAseq、质量细胞术和荧光激活细胞分选。我们还将利用数字空间 ID2调节肿瘤的转录组学(DSP)和在配对的人LGG/HGG样本中验证ID2信号 剖析髓系、NK、T细胞标记物和检查点分子的表达，以阐明ROI对 Mt.Mt.这些结果将阐明ID2诱导BMDM细胞分化为亲肿瘤髓系细胞的机制。目标2. 确定CD74/巨噬细胞移动抑制因子(MIF)轴是否调节BMDM细胞分化 在LGG恶变过程中。我们将通过药物治疗来研究CD74的S在MT中的作用 或者将野生型或CD74KD/KO骨髓细胞移植到小鼠RCAS/TV--一种胶质瘤模型中。我们将分析 外周血和脑胶质瘤TME用scRNAseq、质量细胞仪和FACS进行检测。我们将通过以下方式验证调查结果 Aya患者成对LGG/HGG的批量RNA测序数据并研究CD74/MIF信号转导途径 驱动T细胞和NK细胞减少，从而支持LGG MT。我们还将在CD74/MIF调制中利用DSP 并在配对的人LGG/HGG样本中验证CD74信号，以剖析髓系细胞的表达， NK、T细胞标志物和检查点分子，以阐明对MT至关重要的ROI，这将表明潜在的 有针对性的漏洞。总体影响：我们的研究将阐明可能使我们能够翻译的新见解 进入增强的免疫监测方法，以延迟和/或预防Aya LGG患者的MT。

Project Summary Low-grade gliomas (LGGs), a heterogeneous group of primary central nervous system tumors, are one of the most common solid tumors in the adolescent and young adult (AYA) population. LGGs can progress to high- grade gliomas (HGGs) via a process known as malignant transformation (MT), resulting in dismal prognoses. The mechanisms driving MT of LGG remain poorly understood. In contrast to LGGs, we and others have shown that HGGs have increased levels of bone marrow-derived myeloid cells (BMDMs) and myeloid-derived suppressor cells (MDSCs) in peripheral circulation, with an intra-tumoral enrichment of tumor-associated macrophages (TAMs) and a paucity of CD8+T and natural killer (NK) cells. Although myeloid cells are known to accumulate during glioma progression, it remains unclear if these cells have a causal role in driving MT. Central hypothesis: Reductions in anti-tumor reactive CD8+T and NK cell-dependent immune surveillance are responsible for LGG to HGG malignant transformation in AYA patients and these reductions are dictated by increased infiltration and immune suppressive activity of tumor infiltrating BMDMs. We have identified two independent myeloid associated pathways that are responsible for these BMDM phenotypes and we propose to test the central hypothesis through the following two aims: Aim 1. Delineate the dependency of BMDM cell differentiation on inhibitor of DNA binding protein 2 (ID2). Using AYA RCAS/tv-a glioma mouse models, we will knockdown ID2 in BMDMs before and during MT. We will examine effects on pro-tumoral myeloid cells and evaluate intra-tumoral T and NK cells and mobilization in blood, spleen, and bone marrow at various times with scRNAseq, mass cytometry, and fluorescent-activated cell sorting. We will also leverage digital spatial transcriptomics (DSP) in ID2 modulated tumors and validate ID2 signaling in human paired LGG /HGG samples to dissect the expression of myeloid, NK, T cell markers, and checkpoint molecules to illuminate ROIs critical to MT. Results will illuminate ID2 mechanisms of BMDM cell differentiation to pro-tumoral myeloid cells. Aim 2. Determine if the CD74/macrophage migration inhibitory factor (MIF) axis regulates BMDM cell differentiation during LGG malignant transformation. We will investigate CD74’s role in MT using pharmacological treatments or transplant wild-type or CD74 KD/KO bone marrow cells in a murine RCAS/tv-a glioma model. We will analyze peripheral blood and the glioma TME using scRNAseq, mass cytometry, and FACS. We will validate findings with bulk RNA sequencing data on paired LGG/HGGs from AYA patients and investigate how CD74/MIF signaling drives reduced T and NK cells which supports LGG MT. We will also leverage DSP in CD74/MIF modulated tumors and validate CD74 signaling in human paired LGG/HGG samples to dissect the expression of myeloid, NK, T cell markers, and checkpoint molecules to illuminate ROIs critical to MT that will indicate potentially targetable vulnerabilities. Overall Impact: Our studies will illuminate novel insights that may enable us to translate into enhanced immune surveillance approaches to delay and/or prevent MT for AYA LGG patients.