Non-inflammatory tumor microenvironment of Diffuse Intrinsic Pontine Glioma (DIPG). Non-inflammatory tumor microenvironment of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal malignancy of the childhood central nervous system, with a median overall survival of 9-11 months. We have previously shown that primary DIPG tissue contains numerous tumor-associated macrophages, and substantial work has demonstrated a significant pathological role for adult glioma-associated macrophages. However, work over the past decade has highlighted many molecular and genomic differences between pediatric and adult glioblastomas (GBM). Thus, we directly compared inflammatory characteristics of DIPG and adult GBM. We found that the leukocyte (CD45+) compartment in primary DIPG tissue samples is predominantly composed of CD11b+ macrophages, with very few CD3+ T-lymphocytes. In contrast, T-lymphocytes are more abundant in adult GBM tissue samples. RNA sequencing of macrophages isolated from primary tumor samples revealed that DIPG- and adult GBM-associated macrophages both express gene programs related to ECM remodeling and angiogenesis, but DIPG-associated macrophages express substantially fewer inflammatory factors than their adult GBM counterparts. Examining the secretome of glioma cells, we found that patient-derived DIPG cell cultures secrete markedly fewer cytokines and chemokines than patient-derived adult GBM cultures. Concordantly, bulk and single-cell RNA sequencing data indicates low to absent expression of chemokines and cytokines in DIPG. Together, these observations suggest that the inflammatory milieu of the DIPG tumor microenvironment is fundamentally different than adult GBM. The low intrinsic inflammatory signature of DIPG cells may contribute to the lack of lymphocytes and non-inflammatory phenotype of DIPG-associated microglia/macrophages. Understanding the glioma subtype-specific inflammatory milieu may inform the design and application of immunotherapy-based treatments. Overall design: RNA-seq of primary isolated microglia/macrophages from early post-mortem DIPG tissue samples, pediatric normal cortex, and adult GBM tissue samples. Libraries were sequenced on Illumina NextSeq 500, 1x75.

弥漫内生型桥脑胶质瘤（DIPG，Diffuse intrinsic pontine glioma）是一种预后极差的儿童中枢神经系统恶性肿瘤，中位总生存期仅9~11个月。本团队既往研究表明，原发性DIPG组织中存在大量肿瘤相关巨噬细胞；既往大量研究也证实，成人胶质瘤相关巨噬细胞在疾病进程中发挥关键病理作用。然而，近十年的研究揭示了儿童与成人胶质母细胞瘤（GBM，glioblastomas）在分子与基因组层面存在诸多差异。因此，本研究直接对比了DIPG与成人GBM的炎症特征。研究发现，原发性DIPG组织样本中的白细胞（CD45+）群主要由CD11b+巨噬细胞构成，CD3+ T淋巴细胞占比极低。与之相反，成人GBM组织样本中的T淋巴细胞丰度更高。对原发性肿瘤样本中分离的巨噬细胞进行RNA测序结果显示，DIPG相关巨噬细胞与成人GBM相关巨噬细胞均表达与细胞外基质（ECM，extracellular matrix）重塑、血管生成相关的基因程序，但DIPG相关巨噬细胞的炎症因子表达水平显著低于成人GBM相关巨噬细胞。对胶质瘤细胞分泌组的分析显示，患者来源的DIPG细胞培养液所分泌的细胞因子与趋化因子水平显著低于患者来源的成人GBM细胞培养液。与之一致的是，批量RNA测序（bulk RNA sequencing）与单细胞RNA测序数据均表明，DIPG组织中趋化因子与细胞因子的表达水平极低甚至缺失。综上，上述结果表明DIPG肿瘤微环境的炎症微环境与成人GBM存在本质差异。DIPG细胞本身较低的炎症特征，可能是导致肿瘤微环境中淋巴细胞缺失以及DIPG相关小胶质细胞/巨噬细胞呈非炎症表型的原因。深入了解胶质瘤亚型特异性的炎症微环境，可为免疫治疗策略的设计与应用提供参考依据。实验整体设计：对早期尸检获取的DIPG组织样本、儿童正常皮层组织样本以及成人GBM组织样本中分离的原发性小胶质细胞/巨噬细胞进行RNA测序；测序文库在Illumina NextSeq 500平台上以1×75 bp模式完成测序。