联合米托蒽醌和抗 TGFβ 治疗与 PD-1 阻断通过重塑神经母细胞瘤的肿瘤免疫景观增强抗肿瘤免疫。

BACKGROUND: Poor infiltration of functioning T cells renders tumors unresponsive to checkpoint-blocking immunotherapies. Here, we identified a combinatorial in situ immunomodulation strategy based on the administration of selected immunogenic drugs and immunotherapy to sensitize poorly T-cell-infiltrated neuroblastoma (NB) to the host antitumor immune response. METHODS: 975A2 and 9464D NB cell lines derived from spontaneous tumors of TH-MYCN transgenic mice were employed to study drug combinations able of enhancing the antitumor immune response using in vivo and ex vivo approaches. Migration of immune cells towards drug-treated murine-derived organotypic tumor spheroids (MDOTS) were assessed by microfluidic devices. Activation status of immune cells co-cultured with drug-treated MDOTS was evaluated by flow cytometry analysis. The effect of drug treatment on the immune content of subcutaneous or orthotopic tumors was comprehensively analyzed by flow-cytometry, immunohistochemistry and multiplex immunofluorescence. The chemokine array assay was used to detect soluble factors released into the tumor microenvironment. Patient-derived organotypic tumor spheroids (PDOTS) were generated from human NB specimens. Migration and activation status of autologous immune cells to drug-treated PDOTS were performed. RESULTS: We found that treatment with low-doses of mitoxantrone (MTX) recalled immune cells and promoted CD8 T and NK cell activation in MDOTS when combined with TGFβ and PD-1 blockade. This combined immunotherapy strategy curbed NB growth resulting in the enrichment of a variety of both lymphoid and myeloid immune cells, especially intratumoral dendritic cells (DC) and IFNγ- and granzyme B-expressing CD8 T cells and NK cells. A concomitant production of inflammatory chemokines involved in remodelling the tumor immune landscape was also detected. Interestingly, this treatment induced immune cell recruitment against PDOTS and activation of CD8 T cells and NK cells. CONCLUSIONS: Combined treatment with low-dose of MTX and anti-TGFβ treatment with PD-1 blockade improves antitumor immunity by remodelling the tumor immune landscape and overcoming the immunosuppressive microenvironment of aggressive NB.

背景：功能 T 细胞浸润不良导致肿瘤对检查点阻断免疫疗法无反应。在这里，我们确定了一种基于选择免疫原性药物和免疫疗法给药的组合原位免疫调节策略，以使浸润不良的神经母细胞瘤（NB）对宿主抗肿瘤免疫反应敏感。

方法：使用源自 TH-MYCN 转基因小鼠自发肿瘤的 975A2 和 9464D NB 细胞系，通过体内和体外方法研究能够增强抗肿瘤免疫反应的药物组合。通过微流控装置评估免疫细胞向药物处理的鼠源性器官型肿瘤球体（MDOTS）的迁移。通过流式细胞术分析评估与药物处理的 MDOTS 共培养的免疫细胞的激活状态。通过流式细胞术、免疫组织化学和多重免疫荧光全面分析药物处理对皮下或原位肿瘤免疫含量的影响。使用趋化因子阵列分析检测释放到肿瘤微环境中的可溶性因子。从人 NB 标本中生成患者来源的器官型肿瘤球体（PDOTS）。对药物处理的 PDOTS 中自体免疫细胞的迁移和激活状态进行了研究。

结果：我们发现，当与 TGFβ 和 PD-1 阻断联合使用时，低剂量米托蒽醌（MTX）处理可召回免疫细胞并促进 MDOTS 中 CD8 T 和 NK 细胞的激活。这种联合免疫治疗策略抑制了 NB 的生长，导致多种淋巴和髓样免疫细胞的富集，特别是肿瘤内树突状细胞（DC）和 IFNγ 和 granzyme B 表达的 CD8 T 细胞和 NK 细胞。还检测到涉及重塑肿瘤免疫景观的炎症趋化因子的同时产生。有趣的是，这种治疗方法诱导了针对 PDOTS 的免疫细胞募集和 CD8 T 细胞和 NK 细胞的激活。

结论：低剂量 MTX 联合抗 TGFβ 治疗与 PD-1 阻断可通过重塑肿瘤免疫景观和克服侵袭性 NB 的免疫抑制微环境来改善抗肿瘤免疫。