Immunology Area, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
Pathology Department, IRCCS Sacro Cuore Don Calabria, Negrar, Verona, Veneto, Italy.
J Immunother Cancer. 2021 Jan;9(1). doi: 10.1136/jitc-2020-001313.
Neuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal elements recruited or generated by the cancer cells favor the development of an immune-suppressive microenvironment. Herein, we investigated in NB cell lines and in NB biopsies, the presence of cancer cells with mesenchymal phenotype and determined the immune-suppressive properties of these tumor cells on natural killer (NK) cells.
We assessed the mesenchymal stromal cell (MSC)-like phenotype and function of five human NB cell lines and the presence of this particular subset of neuroblasts in NB biopsies using flow-cytometry, immunohistochemistry, RT-qPCR, cytotoxicity assays, western blot and silencing strategy. We corroborated our data consulting a public gene-expression dataset.
Two NB cell lines, SK-N-AS and SK-N-BE(2)C, exhibited an unprecedented MSC phenotype (CD105/CD90/CD73/CD29/CD146/GD2/TAZ). In these NB-MSCs, the ectoenzyme CD73 and the oncogenic/immune-regulatory transcriptional coactivator TAZ were peculiar markers. Their MSC-like nature was confirmed by their adipogenic and osteogenic differentiation potential. Immunohistochemical analysis confirmed the presence of neuroblasts with MSC phenotype (CD105/CD73/TAZ). Moreover, a public gene-expression dataset revealed that, in stage IV NB, a higher expression of TAZ and CD105 strongly correlated with a poorer outcome.Among the NB-cell lines analyzed, only NB-MSCs exhibited multifactorial resistance to NK-mediated lysis, inhibition of activating NK receptors, signal adaptors and of NK-cell cytotoxicity through cell-cell contact mediated mechanisms. The latter property was controlled partially by TAZ, since its silencing in NB cells efficiently rescued NK-cell cytotoxic activity, while its overexpression induced opposite effects in non-NB-MSC cells.
We identified a novel NB immunoregulatory subset that: (i) displayed phenotypic and functional properties of MSC, (ii) mediated multifactorial resistance to NK-cell-induced killing and (iii) efficiently inhibited, in coculture, the cytotoxic activity of NK cells against target cells through a TAZ-dependent mechanism. These findings indicate that targeting novel cellular and molecular components may disrupt the immunomodulatory milieu of the NB microenvironment ameliorating the response to conventional treatments as well as to advanced immunotherapeutic approaches, including adoptive transfer of NK cells and chimeric antigen receptor T or NK cells.
神经母细胞瘤（NB）是最常见的儿童颅外实体瘤，起源于神经嵴祖细胞，是儿科患者死亡的主要原因。在实体肿瘤中，癌细胞募集或产生的基质成分有利于形成免疫抑制的微环境。在此，我们研究了 NB 细胞系和 NB 活检组织中具有间充质表型的癌细胞，并确定了这些肿瘤细胞对自然杀伤（NK）细胞的免疫抑制特性。
我们使用流式细胞术、免疫组织化学、RT-qPCR、细胞毒性测定、Western blot 和沉默策略评估了五个人类 NB 细胞系的间充质基质细胞（MSC）样表型和功能，以及 NB 活检组织中这种特定的神经母细胞亚群的存在。我们使用公共基因表达数据集来验证我们的数据。
两种 NB 细胞系 SK-N-AS 和 SK-N-BE(2)C 表现出前所未有的 MSC 表型（CD105/CD90/CD73/CD29/CD146/GD2/TAZ）。在这些 NB-MSCs 中，外切酶 CD73 和致癌/免疫调节转录共激活因子 TAZ 是特殊的标记物。它们的 MSC 样性质通过它们的成脂和成骨分化潜能得到证实。免疫组织化学分析证实了具有 MSC 表型（CD105/CD73/TAZ）的神经母细胞的存在。此外，一个公共基因表达数据集显示，在 IV 期 NB 中，TAZ 和 CD105 的高表达与更差的预后强烈相关。在分析的 NB 细胞系中，只有 NB-MSCs 表现出对 NK 介导的裂解、激活 NK 受体抑制、信号接头和通过细胞-细胞接触介导机制的 NK 细胞细胞毒性的多因素耐药性。后一种特性部分受到 TAZ 的控制，因为在 NB 细胞中沉默 TAZ 可有效地恢复 NK 细胞的细胞毒性活性，而其在非 NB-MSC 细胞中的过表达则诱导相反的效应。
我们鉴定了一种新的 NB 免疫调节亚群，该亚群：（i）表现出 MSC 的表型和功能特性，（ii）介导对 NK 细胞诱导杀伤的多因素耐药性，（iii）通过 TAZ 依赖性机制在共培养中有效抑制 NK 细胞对靶细胞的细胞毒性。这些发现表明，靶向新的细胞和分子成分可能破坏 NB 微环境的免疫调节环境，改善对常规治疗以及先进免疫治疗方法（包括 NK 细胞的过继转移和嵌合抗原受体 T 或 NK 细胞）的反应。
