PO.TB10.01 · 肿瘤生物学
丛状神经纤维瘤形成的两步机制：NF-κB通路在神经纤维瘤形成中的作用
Two-step mechanism of plexiform neurofibroma formation: Role of the NF-κB pathway in neurofibroma formation
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
原理：NF1-/- Schwann细胞是丛状神经纤维瘤（PNF）的起源细胞。在该疾病的小鼠模型中，这种良性肿瘤的形成发生在Nf1丢失很久之后，提示Schwann细胞在肿瘤形成过程中经历了继发性改变。然而，在该肿瘤类型中并未观察到额外的遗传学打击。单细胞RNA测序提示，NF-κB信号在已形成的肿瘤中上调。与NF-κB信号是驱动肿瘤信号这一观点一致，在人和小鼠神经纤维瘤中，p-65在神经纤维瘤细胞中呈核内（活化）状态，其中一些是Schwann细胞（Kershner等，2022）。
方法：为检验NF-κB信号的激活是否是神经纤维瘤形成的第二步，我们在肿瘤发展过程中联合运用了多重抗体染色、流式细胞术和RNA测序。体外实验用于确定NF-κB通路调节对Nf1-/- Schwann细胞的影响。最后，我们检验了体内阻断IKK2活性是否能减少肿瘤形成或生长。
结果：我们通过多重成像和流式细胞术在PNF中鉴定出Schwann细胞、成纤维细胞和免疫细胞的标志物。肿瘤Schwann细胞（而非同一小鼠中的肿瘤前Schwann细胞）表达细胞表面标志物CD44和CD49f；培养的Nf1-/- Schwann细胞在暴露于已知激活NF-κB信号的应激源（包括长时间血清剥夺、Poly I:C、IL1beta和TNFalpha）时，或在感染激活型IKK2（其激活NF-κB通路）时，上调这些标志物和核内（活化）p65；这些Schwann细胞增加了作为免疫细胞趋化剂的细胞因子的分泌。在体内和体外均观察到EMT基因的同步增加。用NF-κB通路抑制剂BAY 11-7082联合MEK抑制剂Mirdametinib处理DhhCre;Nf1fl/fl小鼠，减少了CD44+ CD49f+ Schwann细胞群、肿瘤细胞增殖、肿瘤免疫细胞群和肿瘤细胞因子。
结论：我们提出PNF形成的两步过程，即Schwann细胞中的Nf1丢失为起始步骤，通过NF-κB通路激活和Schwann细胞重编程形成炎症微环境为第二步。（由DOD-HT9425-1-0435（授予NR和JS）、NIH NS115438R01（授予DAL和NR）以及儿童肿瘤基金会青年研究者奖（授予RR）资助）
查看英文原文 English abstract
Rationale : NF1-/- Schwann cells are the cells of origin in plexiform neurofibroma (PNF). This benign tumor formation occurs long after Nf1 loss in mouse models of the disease, suggesting that Schwann cells undergo secondary changes during tumor formation. However, additional genetic hits are not observed in this tumor type. Single cell RNA-sequencing implicated NF-κB signaling as upregulated in established tumor formation. Consistent with the idea that NF-κB signaling is a tumor driving signal, in human and mouse neurofibromas p-65 was nuclear (active) in neurofibroma cells, some of which were Schwann cells (Kershner et al., 2022).
Methods : To test if activation of NF-κB signaling is a second step in neurofibroma formation we used a combination of multiplexed antibody staining, flow cytometry and RNA sequencing in tumors over their development. In vitro assays were utilized to determine the effect of NF-κB pathway modulation in Nf1-/- Schwann cells. Finally, we tested if blocking IKK2 activity in vivo reduces tumor formation or growth.
Results : We identified markers of Schwann cells, fibroblasts and immune cells in PNF by multiplex imaging and flow cytometry. Tumor Schwann cells, but not pre-tumor Schwann cells in the same mice, expressed the cell surface markers CD44 and CD49f; cultured Nf1-/- Schwann cells upregulate these markers and nuclear (active) p65 when exposed to stressors known to activate NF-κB signaling, including prolonged serum depletion, Poly I:C, IL1beta, and TNFalpha, or when infected with activated IKK2, which activates the NF-κB pathway; these Schwann cells increased secretion of cytokines that are immune cell chemoattracts. Concurrent increases in EMT genes were observed in vivo and in vitro . Treatment of DhhCre;Nf1fl/fl mice with the NF-κB pathway inhibitor BAY 11-7082 combined with MEK inhibitor Mirdametinib reduced the CD44+ CD49f+ Schwann cell population, tumor cell proliferation, the tumor immune cell population, and tumor cytokines.
Conclusion: A two-step process to PNF formation is proposed, with Nf1 loss in Schwann cells an initiating step and the formation of an inflammatory microenvironment via activation of the NF-κB pathway and Schwann cell reprogramming as a second step. (Supported by DOD-HT9425-1-0435 (to NR and JS), NIH NS115438R01 (to DAL and NR) and a Children's Tumor Foundation Young Investigator Award to RR)
利益披露 Disclosure
R. Ravindran, None..
N. Kedei, None..
E. Youn, None..
K. Choi, None..
A. Volz, None..
J. Pundavela, None..
D. A. Largaespada, None..
J. F. Shern, None..
N. Ratner, None.