联合多组学分析和机器学习分析揭示Bregs和RPS2促进鼻咽癌骨转移

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中文摘要

背景： 骨是鼻咽癌（NPC）患者远处转移的主要部位，骨转移（BM）显著增加死亡率。破译癌细胞与其肿瘤微环境（TME）之间复杂的串扰对于确定旨在消除转移起始细胞和预防明显转移形成的治疗靶点至关重要。调节性B细胞（Bregs）是一种具有强大免疫抑制功能的特化B细胞亚群，在TME中发挥关键的调节作用。然而，它们在NPC的BM微环境中的具体贡献、表型特征和潜在分子机制仍未完全表征，有待充分阐明。本研究旨在探讨Bregs在NPC BM背景下的浸润动态、功能影响和分子基础。
方法： 本研究利用公共数据库中的单细胞测序数据和RNA测序（RNA-seq）微阵列矩阵进行分析。随后，结合三种机器学习方法，即最小绝对收缩和选择算子（LASSO）回归分析、随机森林算法和Boruta算法，以识别预后基因。证实了BM患者原发灶中Bregs的高浸润和RPS2的高表达，并构建了相关的预后特征。使用流式细胞术、细胞计数试剂盒-8（CCK-8）测定、迁移测定、Transwell测定和共培养系统评估Bregs对细胞增殖和转移的影响。建立了心内注射诱导的肿瘤移植动物模型，并使用生物发光成像（BLI）、数字放射摄影（DR）和微型计算机断层扫描（Micro-CT）评估Bregs对肿瘤细胞BM的影响。最后，进行临床队列分析以确定Bregs与NPC中BM的相关性。
结果： Bregs在伴有BM的NPC原发灶中表现出高浸润，并且Bregs中RPS2的高表达与预后高度相关。在机制上，RPS2上调Bregs分泌白细胞介素-10（IL-10）。IL-10随后激活PI3K/AKT通路，进而上调肿瘤细胞表面CXCR6的表达。这些肿瘤细胞在骨中巨噬细胞分泌的CXCL16的吸引下，表现出增加的BM倾向。在NPC患者原发灶中，三级淋巴结构内表达RPS2的Bregs与BM高度相关。
结论： Bregs和RPS2可用于NPC中BM的预后评估。靶向Bregs或IL-10的药理学抑制可能是延缓 and 治疗NPC中BM的可行方法。

英文摘要

BACKGROUND: The bone is the primary site of distant metastasis in patients with nasopharyngeal carcinoma (NPC), and bone metastasis (BM) significantly increases mortality. Deciphering the complex crosstalk between cancer cells and their tumor microenvironment (TME) is crucial for identifying therapeutic targets aimed at eliminating metastasis-initiating cells and preventing the establishment of overt metastases. Regulatory B cells (Bregs), a specialized B-cell subset endowed with potent immunosuppressive functions, play a pivotal regulatory role within the TME. However, their specific contributions, phenotypic characteristics, and underlying molecular mechanisms within the BM niche of NPC remain incompletely characterized and await full elucidation. This study aims to investigate the infiltration dynamics, functional impact, and molecular underpinnings of Bregs in the context of NPC BM.
METHODS: The single-cell sequencing data and RNA sequencing (RNA-seq) microarray matrices from public databases were utilized for the analysis in this study. Subsequently, three machine learning methods, least absolute shrinkage and selection operator (LASSO) regression analysis, random forest algorithm, and Boruta algorithm were combined to identify prognostic genes. The high infiltration of Bregs and high expression of RPS2 in the primary lesions of BM patients were confirmed, and relevant prognostic signatures were constructed. Flow cytometry, cell counting kit-8 (CCK-8) assay, migration assay, Transwell assay, and co-culture system were used to evaluate the effects of Bregs on cell proliferation and metastasis. An animal model of intracardiac injection-induced tumor transplantation was established, and bioluminescence imaging (BLI), digital radiography (DR), and micro computed tomography (Micro-CT) were utilized to assess the impact of Bregs on BM of tumor cells. Finally, clinical cohort analysis was conducted to determine the correlation between Bregs and BM in NPC.
RESULTS: Bregs exhibit high infiltration in the primary lesions of NPC with BM, and the high expression of RPS2 in Bregs is highly correlated with prognosis. Mechanistically, RPS2 upregulates the secretion of interleukin-10 (IL-10) by Bregs. IL-10 then activates the PI3K/AKT pathway, which in turn upregulates the expression of the CXCR6 on the surface of tumor cells. These tumor cells, under the attraction of CXCL16 secreted by macrophages in bone, show an increased tendency to undergo BM. In the primary lesions of NPC patients, Bregs expressing RPS2 within tertiary lymphoid structures are highly associated with BM.
CONCLUSIONS: Bregs and RPS2 can be utilized for the prognostic evaluation of BM in NPC. Pharmacological inhibition targeting Bregs or IL-10 may be a feasible approach to delay and treat BM in NPC.