Clinical Significance and Mechanism of Action of Exosomal microRNAs in Neuroblastoma chemoresistance

外泌体 microRNA 在神经母细胞瘤化疗耐药中的临床意义和作用机制

• 批准号: 10478753
• 负责人: Muller Fabbri
• 金额: $ 37.37万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10478753
• 关键词: Address; Affect; Antibodies; Autologous; Back; Binding; Biological; Biology; CASP8 gene; Cells; Chemoresistance; Child; Childhood Solid Neoplasm; Cisplatin; Clinical; Combined Modality Therapy; Data; Down-Regulation; Drug resistance; Engineering; Gene Expression; Gene Expression Regulation; Genes; Goals; Grant; Growth; Hematopoietic Stem Cell Transplantation; Human; Immunotherapy; Impairment; In Vitro; Infiltration; Interleukin-6; Knowledge; MYCN gene; Malignant Childhood Neoplasm; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Mediating; MicroRNAs; Molecular; Molecular Target; Multi-Drug Resistance; Multidrug Resistance Gene; Neoplasm Metastasis; Neuroblastoma; Operative Surgical Procedures; Outcome; Patients; Plasma; Primary Neoplasm; Prognosis; Proteins; Protocols documentation; Publishing; Radiation therapy; Recurrence; Refractory; Relapse; Research; Resistance; Role; Sampling; Series; Solid; Suggestion; TLR8 gene; TNF gene; TP53 gene; Therapeutic; Treatment Protocols; Tretinoin; Tumor Suppressor Proteins; Tumor-associated macrophages; Untranslated RNA; Up-Regulation; base; burden of illness; chemotherapy; clinical outcome measures; clinically significant; cranium; cytokine; drug development; exosome; experimental study; extracellular vesicles; functional loss; high risk; high risk population; improved; in vitro testing; in vivo; macrophage; nanoparticle; nanoparticle delivery; neuroblastoma cell; novel strategies; novel therapeutic intervention; novel therapeutics; patient subsets; radiological imaging; resistance mechanism; standard care; standard of care; therapy resistant; tumor; tumor growth; tumor microenvironment

Neuroblastoma (NB) is the most common solid cancer in children outside of the skull and it still kills about 40% of patients. There is increasing evidence that the tumor microenvironment promotes resistance of NB to chemotherapy. In particular, Tumor-Associated Macrophages (TAMs) promote NB growth and resistance. However, there are two fundamental gaps in our knowledge of this interaction: 1) We do not know which molecular mechanisms mediate TAM pro-tumoral effects and consequently we are unable to exploit such mechanisms for new therapeutic purposes; 2) We have not identified a “systemic” parameter that reflects the degree of TAM infiltration in the primary tumor, and consequently we cannot identify which subsets of patients would particularly benefit from an anti-TAM therapy. Our preliminary data support a role for microRNAs (miRs) within exosomes as responsible for the increased NB proliferation and drug resistance through the direct targeting of TP53, the most frequently dys-regulated gene in human cancers and with a well established role in multi-drug resistance in NB. Specifically, NB cells secrete exosomal miR-21, which is up-taken by surrounding macrophages and can bind to Toll-like receptor 8 (TLR8), triggering TLR8 activation in macrophages. As a consequence of this activation, we showed up- regulation of miR-155, -487a and -597, all predicted to target TP53. We also engineered a nanoparticle coated with anti-CD163 antibody to specifically silence miR-155 in TAMs (that are CD163+). Finally, we were able to develop a modified protocol that successfully isolates purer exosomes (meaning with lower protein contaminants) both from cell supernatants and from patients’ plasma. With this protocol we isolated CD163+ exosomes (released by TAMs) from the plasma of NB patients and healthy donors, and showed increased levels of exosomal miR-155 in the plasma of NB patients compared to healthy donors. Therefore, we hypothesize that NB cells, by secreting exosomal miR-21 that binds to TLR8 in surrounding TAMs, induce the secretion of exosomal miR-155, -487a and -597 by TAMs and these miRs are transferred back to NB cells, where they silence TP53 and increase NB multi-drug resistance. We also hypothesize that targeting these miRs will restore sensitivity to chemotherapy. Finally, we believe that the levels of TAM-derived exosomal miRs will reflect the degree of TAM infiltration in the primary tumor and will correlate with clinical outcome measures. We will investigate these hypotheses in 3 specific aims: 1) a study of the mechanisms by which exosomal miRs induce resistance to therapy in NB; 2) an assessment of the therapeutic potential of targeting NB and TAM-derived exosomal miRs to overcome NB resistance; 3) a determination of TAM-derived exosomal miRs as indicators of TAM-infiltration in the primary tumor and of clinical outcome measures. The successful completion of this research will identify new molecular targets for NB, identify subsets of patients who can benefit from an anti-TAM therapy, and will increase the number of saved lives of children affected by NB.

神经母细胞瘤(NB)是儿童颅外最常见的实体癌，其致死率约为40%。 病人的数量。越来越多的证据表明，肿瘤微环境促进了对NB的耐药性 化疗。特别是，肿瘤相关巨噬细胞(TAMs)促进了NB的生长和耐药。 然而，我们对这种相互作用的了解存在两个基本差距：1)我们不知道是哪一个 促肿瘤作用的分子机制，因此我们无法利用这种机制 新的治疗目的的机制；2)我们还没有确定一个反映 在原发肿瘤中的浸润程度，因此我们无法确定哪些患者亚群 尤其是从抗疗法中受益。 我们的初步数据支持外体中的microRNAs(MiRs)的作用是导致 通过直接靶向最常受Dys调控的TP53来实现NB的增殖和耐药性 该基因在人类癌症中存在，并且在NB的多药耐药中起着公认的作用。具体地说，NB细胞 分泌外体miR-21，可被周围巨噬细胞摄取并与Toll样受体8结合 (TLR8)，触发巨噬细胞中的TLR8激活。作为这次激活的结果，我们出现了- MiR-155、-487a和-597的调节，都预测以TP53为靶点。我们还设计了一种纳米粒子涂层 用抗CD163抗体特异性沉默TAMs中的miR-155(即CD163+)。最后，我们终于能够 开发一种改进的方案，成功地分离出更纯的外切体(即较低的蛋白质 污染物)来自细胞上清液和患者血浆。通过这个方案，我们分离出了CD163+ 从NB患者和健康献血者血浆中释放的Exosome(由TAMS释放)，并显示增加 NB患者与健康献血员血浆外体miR-155水平的比较因此，我们 假设NB细胞通过分泌外体miR-21与周围TAMs中的TLR8结合，诱导 TAMS分泌外体miR-155、-487a和-597，这些miR被转移回NB细胞， 在那里，它们抑制TP53并增加NB的多药耐药性。我们还假设以这些为目标 MIRS将恢复对化疗的敏感性。最后，我们认为来源的外体MIR的水平 将反映在原发灶肿瘤中的侵袭程度，并与临床预后指标相关。 我们将在三个具体目标上研究这些假说：1)研究外切体的作用机制 MIR导致对NB的治疗抵抗；2)靶向NB和 衍生的胞外体miRs克服Nb耐药；3)衍生的胞外体miRs的测定 作为在原发肿瘤中的浸润性指标和临床预后指标。成功者 这项研究的完成将确定NB的新分子靶点，确定可以 受益于抗疗法，并将增加受NB影响的儿童的获救生命。

项目成果

MicroRNA-16 Restores Sensitivity to Tyrosine Kinase Inhibitors and Outperforms MEK Inhibitors in KRAS-Mutated Non-Small Cell Lung Cancer.

• DOI: 10.3390/ijms222413357
• 发表时间: 2021-12-12
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Fanini F;Bandini E;Plousiou M;Carloni S;Wise P;Neviani P;Murtadha M;Foca F;Fabbri F;Vannini I;Fabbri M
• 通讯作者: Fabbri M

mRNA and miRNA Profiles of Exosomes from Cultured Tumor Cells Reveal Biomarkers Specific for HPV16-Positive and HPV16-Negative Head and Neck Cancer.

• DOI: 10.3390/ijms21228570
• 发表时间: 2020-11-13
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Ludwig S;Sharma P;Wise P;Sposto R;Hollingshead D;Lamb J;Lang S;Fabbri M;Whiteside TL
• 通讯作者: Whiteside TL

Overexpression of ultraconserved region 83- induces lung cancer tumorigenesis.

• DOI: 10.1371/journal.pone.0261464
• 发表时间: 2022
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Vannini I;Ferracin M;Fabbri F;Fabbri M
• 通讯作者: Fabbri M