肿瘤细胞治疗研究
英文原题：Transition to a mesenchymal state in neuroblastoma may be characterized by a high expression of GD2 and by the acquisition of immune escape from NK cells.
神经母细胞瘤是一种异质性肿瘤，是神经母细胞瘤免疫治疗中的又一障碍。
背景：神经母细胞瘤（NB）包括肾上腺素能（ADRN）和未分化间充质（MES）亚群。含唾液酸的神经节苷脂糖鞘脂 GD2 在神经外胚层来源肿瘤中广泛过表达，促进恶性表型。治疗后和复发肿瘤中 MES 细胞显著富集，且 GD2 表达降低，可能导致 GD2 靶向免疫治疗失败。NK 细胞是可有效杀伤肿瘤的关键先天免疫细胞亚群，但包括肿瘤细胞和肿瘤相关细胞在内的肿瘤微环境（TME）可能抑制其效应功能。 方法：研究分析了 8 份 NB 原代培养物；与商业细胞系相比，它们更忠实地反映肿瘤细胞特征。研究了 4 份原代 NB-MES 细胞培养物，以及来自同一患者的两组 MES/ADRN 配对原代培养物（691 和 717）。尤其在 6 份人 NB 原代培养物中，采用流式细胞术、RT-qPCR 和细胞毒实验，评估其表型、GD2 表达及调控 GD2 表达的酶，并研究其与 NK 细胞的相互作用。 结果：研究鉴定出成熟型（CD105+/CD133−）和未分化型（CD133+/CD105−）NB 亚群，均高表达 MES 转录本 WWTR1 和 SIX4。未分化 MES 细胞对 NK 介导的杀伤具有强耐受性；成熟 NB-MES 细胞耐受性中等，并可对 NK 细胞产生一定免疫调节作用，但不抑制其细胞溶解活性。值得注意的是，无论未分化或成熟表型如何，NB-MES 细胞均表达 GD2；与 NK 细胞共培养后，未分化 NB-MES 细胞 GD2 可进一步上调，形成 GD2 高表达的成熟间充质神经母细胞。691 和 717 细胞均高表达 GD2，且耐受 NK 细胞介导杀伤；临床使用的抗 GD2 单克隆抗体 dinutuximab beta 可克服这种耐受。 结论：NB 是一种异质性肿瘤，为免疫治疗带来额外障碍。但与商业 NB 细胞的既往报道不同，且不受 MES/ADRN 表型影响，GD2 表达及其对抗 GD2 单克隆抗体介导抗体依赖性细胞毒作用（ADCC）的敏感性，提示抗 GD2 免疫疗法可能有效。
BACKGROUND: Neuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function. METHODS: We studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays. RESULTS: We identified mature (CD105 + /CD133 - ) and undifferentiated (CD133 + /CD105 - ) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2 bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic. CONCLUSIONS: NB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.
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