PO.TB10.15 · 肿瘤生物学
RD3缺陷决定的外泌体遗传货物转变引导神经母细胞瘤的进展性演变
Exosomal genetic cargo shift dictated by RD3 deficiency steers progressive neuroblastoma evolution
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
外泌体是全身性细胞通讯的核心介质，通过遗传和分子货物的动态交换引导肿瘤演变。神经母细胞瘤（NB）是一种起源于胚胎的致命性儿童肿瘤，其中治疗抵抗性癌症干细胞克隆的选择和富集具有决定性影响。近期，我们发现获得性RD3缺陷促使肿瘤细胞可塑性并调控肿瘤进展。在此，我们研究了RD3是否控制外泌体遗传货物的递送。为此，我们利用了自定义存档的神经嵴细胞特异性Cre条件性LoxP导向RD3敲除（KO，转基因小鼠）驱动的人类同线自发性NB。从RD3-KO小鼠分离血清外泌体，并与其野生型对应物进行比较。使用Total Exosome RNA and Protein Isolation试剂盒分离外泌体RNA，通过纳米颗粒追踪分析、流式细胞术、透射电子显微镜进行表征，然后通过双端Illumina RNA测序进行分析。差异基因表达（DEG）采用log₂倍数变化及log₂ FDR计算。DEG分析鉴定出一个与动物个体无关的特征，包含53个上调基因和另外1,396个下调基因。Ingenuity Pathway Analysis中的功能注释揭示了治疗抵抗通路的富集，包括VEGF的血管生成信号（VEGFA、KDR、FLT1）、NAD⁺补救途径II介导的DNA损伤反应（NMRK1、NT5C2、NT5C1A）、细胞周期控制（CDK6、CDC7、SIN3A）、促生存CXCR4信号（HSP90AA1、RAB11FIP3）、细胞外基质重塑（COL1A1、ADAM12、HSPG2、CD31）以及免疫/炎症信号（CD27、IL-1）通路。此外，包括调控迁移、侵袭的RhoGDI信号（CDC42、PAK1、ARHGEF2）以及调控干性维持的WNT/beta-catenin信号（SOX17、MAP3K7、WNT9A）在内的致癌细胞信号通路被决定性地重编程。这些结果首次明确证明，RD3缺陷驱动促进治疗抵抗的分子重排，突显其在能够耐受强化多模式临床治疗的外泌体介导癌细胞信号中的核心功能。至关重要的是，我们的研究结果将RD3调控的外泌体遗传货物定位为一种有前景的生物标志物和治疗靶点，以更好地临床管理进展性NB。资助：DOD-CA-210339；OCAST-HR19-04；NIH-P20GM103639
查看英文原文 English abstract
Exosomes are central mediators of systemic cellular communication, directing tumor evolution through the dynamic exchange of genetic and molecular cargo. Neuroblastoma (NB), a lethal pediatric tumor of embryonal origin, where selection and enrichment of treatment-resistant cancer stem cell clones have decisive impact. Recently, we identified acquired RD3 deficiency prompts tumor cell plasticity and regulates tumor progression. Herein, we investigated whether RD3 controls delivery of exosomal genetic cargo. For this we utilized our custom archived Neural crest cell specific Cre conditional LoxP-directed RD3-Knockout (KO, Tg mice) driven human syntenic spontaneous NB. Serum exosomes were isolated, from RD3-KO mice and compared with their wild-type counterparts. Exosomal RNA was isolated using the Total Exosome RNA and Protein Isolation kit, characterized by nanoparticle tracking analysis, flow cytometry, transmission electron microscopy, and was then analyzed by paired-end Illumina RNA sequencing. Differential gene expression (DEG) was computed using log₂ fold change with log₂ FDR. DEG analysis identified an animal-independent signature of 53 upregulated and another 1,396 downregulated genes. Functional annotation in Ingenuity Pathway Analysis revealed enrichment in therapy-resistance pathways, including angiogenesis signaling by VEGF ( VEGFA, KDR, FLT1 ), NAD⁺ salvage pathway II mediated DNA damage response ( NMRK1, NT5C2, NT5C1A ), cell cycle control ( CDK6, CDC7, SIN3A ), pro-survival CXCR4 signaling ( HSP90AA1, RAB11FIP3 ), extracellular matrix remodeling ( COL1A1, ADAM12, HSPG2, CD31 ), and immune/inflammatory signaling (CD27, IL-1) pathways. Furthermore, oncogenic cell signaling pathways, including RhoGDI signaling ( CDC42, PAK1, ARHGEF2), regulating migration, invasion, and WNT/beta-catenin signaling ( SOX17, MAP3K7, WNT9A ), regulating stemness maintenance, were decisively reprogrammed. These results, for the first time, distinctly demonstrate that RD3 deficiency drives molecular rearrangements that promote therapy resistance, underscoring its central function in exosome-mediated cancer cell signaling that survives intensive multimodal clinical therapy. Crucially, our findings position RD3-regulated exosomal genetic cargo as a promising biomarker and therapeutic target for better clinical management of progressive NB. FUNDING: DOD-CA-210339; OCAST-HR19-04; NIH-P20GM103639
利益披露 Disclosure
A. Jahir Hussain, None..
S. Mohanvelu, None..
L. Periyasamy, None..
P. Subramanian, None..
S. Aravindan, None..
N. Aravindan, None.