PO.TB05.01 · 肿瘤生物学
通过空间RNA与蛋白分析解析颅内生殖细胞肿瘤的复杂生态系统
Dissecting the complex ecosystem of intracranial germ cell tumors by spatial RNA and protein analysis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景与目的：中枢神经系统生殖细胞瘤的特征是大量免疫细胞浸润。这种炎症成分可能极为广泛，以至于因掩盖恶性生殖细胞肿瘤（GCT）成分而给肿瘤诊断带来挑战。同样地，在混合性生殖细胞肿瘤中，往往难以检测出决定其生物学行为和治疗分层的其他恶性成分。在本研究中，我们采用了近期的综合性技术，以研究肿瘤微环境（TME）中恶性成分以及炎症成分的组成和空间分布。
方法：我们通过对福尔马林固定、石蜡包埋（FFPE）肿瘤样本进行免疫表型分析和图像分析，系统性地表征了一组生殖细胞瘤队列中的免疫细胞。采用Nanostring技术生成总体mRNA表达数据，采用Visium-HD技术生成高分辨率空间RNA表达数据。用后一种方法我们研究了10例生殖细胞瘤和3例混合性生殖细胞肿瘤。通过原位RNA杂交和免疫组织化学对个别候选转录本和蛋白进行了验证。
结果：通过Visium-HD进行的空间转录组学可灵敏地检测出不同的恶性生殖细胞肿瘤成分，包括生殖细胞瘤、卵黄囊瘤、胚胎性癌和绒毛膜癌成分，以及免疫细胞的空间分布。生殖细胞瘤中的肿瘤浸润淋巴细胞数量丰富，以CD3阳性T细胞为主，包括CD4阳性辅助性T细胞、CD8阳性细胞毒性T细胞以及散在分布的FoxP3阳性调节性T细胞。B淋巴细胞数量多于浆细胞。部分生殖细胞瘤和混合性GCT表现出三级淋巴结构。肿瘤相关巨噬细胞（TAM）由活化的PD-L1阳性巨噬细胞簇和散在分布的表达CD163的抗炎性巨噬细胞组成。虽然生殖细胞瘤细胞在RNA和蛋白水平上均不表达PD-L1，但鉴定出作为抗凋亡基因的BIRC5（survivin）的高表达。空间mRNA表达分析提示恶性GCT细胞与TME成分之间存在特异性配体-受体相互作用。特别是，生殖细胞瘤细胞产生高水平的巨噬细胞迁移抑制因子（MIF），并且发现其受体CD74在邻近的炎症细胞中表达。
结论：总之，空间高分辨率转录组学能够根据其特征谱灵敏地识别不同的恶性生殖细胞肿瘤成分。在生殖细胞瘤中观察到的强烈免疫反应涉及多种炎症性以及抑制性机制。GCT与TME细胞之间的相互作用分析提示，在颅内GCT细胞的生长和存活调控中存在旁分泌机制。
查看英文原文 English abstract
Background and Objectives: Central nervous system germinomas are characterized by a massive immune cell infiltrate. This inflammatory component can be so extensive that tumor diagnostics is challenging because of masking of the malignant germ cell tumor (GCT) component. Similarely, it is often difficult to detect other malignant components in mixed germ cell tumors that dictate the biology and treatment stratification. In this study we employed recent comprehensive technologies to study the composition and spatial distribution of malignant as well as inflammatory components of the tumor microenvironment (TME).
Methods: We systematically characterized immune cells in a cohort of germinomas by immunophenotyping and image analysis of formalin-fixed, paraffin-embedded (FFPE) tumor samples. Bulk mRNA expression data was generated by Nanostring technology and high-resolution spatial RNA expression data by Visium-HD technology. With the latter method we studied 10 germinomas and 3 mixed germ cell tumors. Individual candidate transcripts and proteins were validated by in-situ RNA hybridization and immunohistochemistry.
Results: Spatial transcriptomics by Visium-HD allowed the sensitive detection of different malignant germ cell tumor components including germinoma, yolk sac tumor, embryonal carcinoma and choriocarcinoma components, as well as the spatial distribution of immune cells. Tumor infiltrating lymphocytes in germinomas were abundant and predominated by CD3-positive T cells, including CD4-positive T-helper cells, CD8-positive cytotoxic T cells and interspersed FoxP3-positive regulatory T cells. B lymphocytes outnumbered plasma cells. Some germinomas and mixed GCT showed tertiary lymphoid structures. Tumor-associated macrophages (TAM) consisted of clusters of activated PD-L1-positive macrophages and interspersed anti-inflammatory macrophages expressing CD163. While germinoma cells did not express PD-L1 on both the RNA and protein level, a high expression of BIRC5 (survivin) as anti-apoptotic gene was identified. Spatial mRNA expression analysis indicated specific ligand-receptor interactions between malignant GCT cells and TME components. In particular, germinoma cells produced high levels of macrophage migration inhibitory factor (MIF), and its receptor CD74 was found expressed in adjacent inflammatory cells.
Conclusions: In conclusion, spatial high resolution transcriptomics was able to sensitively identify different malignant germ cell tumor components by their signature. The strong immune reaction observed in germinomas involved various inflammatory as well as suppressive mechanisms. Interaction analysis between GCT and TME cells indicated paracrine mechanisms in the control of growth and survival of intracranial GCT cells.
利益披露 Disclosure
T. Pietsch, None..
V. Dreschmann, None..
P. Zapka, None..
E. Dörner, None..
C. Vokuhl, None.