Li Yutao, Sharma Amit, Wu Xiaolong, Weiher Hans, Skowasch Dirk, Essler Markus, Schmidt-Wolf Ingo G H
Department of Integrated Oncology, Center for Integrated Oncology (CIO) Bonn, University Hospital Bonn, Bonn, Germany.
Department of Neurosurgery, University Hospital Bonn, Bonn, Germany.
Front Oncol. 2022 May 11;12:713476. doi: 10.3389/fonc.2022.713476. eCollection 2022.
Cancer heterogeneity poses a serious challenge concerning the toxicity and adverse effects of therapeutic inhibitors, especially when it comes to combinatorial therapies that involve multiple targeted inhibitors. In particular, in non-small cell lung cancer (NSCLC), a number of studies have reported synergistic effects of drug combinations in the preclinical models, while they were only partially successful in the clinical setup, suggesting those alternative clinical strategies (with genetic background and immune response) should be considered. Herein, we investigated the antitumor effect of cytokine-induced killer (CIK) cells in combination with ALK and PD-1 inhibitors on genetically variable NSCLC cell lines.
We co-cultured the three genetically different NSCLC cell lines NCI-H2228 (EML4-ALK), A549 (KRAS mutation), and HCC-78 (ROS1 rearrangement) with and without nivolumab (PD-1 inhibitor) and crizotinib (ALK inhibitor). Additionally, we profiled the variability of surface expression multiple immune checkpoints, the concentration of absolute dead cells, intracellular granzyme B on CIK cells using flow cytometry as well as RT-qPCR. ELISA and Western blot were performed to verify the activation of CIK cells.
Our analysis showed that (a) nivolumab significantly weakened PD-1 surface expression on CIK cells without impacting other immune checkpoints or PD-1 mRNA expression, (b) this combination strategy showed an effective response on cell viability, IFN-γ production, and intracellular release of granzyme B in CD3 CD56 CIK cells, but solely in NCI-H2228, (c) the intrinsic expression of Fas ligand (FasL) as a T-cell activation marker in CIK cells was upregulated by this additive effect, and (d) nivolumab induced Foxp3 expression in CD4CD25 subpopulation of CIK cells significantly increased. Taken together, we could show that CIK cells in combination with crizotinib and nivolumab can enhance the anti-tumor immune response through FasL activation, leading to increased IFN-γ and granzyme B, but only in NCI-H2228 cells with EML4-ALK rearrangement. Therefore, we hypothesize that CIK therapy may be a potential alternative in NSCLC patients harboring EML4-ALK rearrangement, in addition, we support the idea that combination therapies offer significant potential when they are optimized on a patient-by-patient basis.
癌症异质性给治疗性抑制剂的毒性和不良反应带来了严峻挑战，尤其是在涉及多种靶向抑制剂的联合治疗中。特别是在非小细胞肺癌（NSCLC）中，多项研究报告了药物组合在临床前模型中的协同效应，但在临床应用中仅取得了部分成功，这表明应考虑其他临床策略（结合基因背景和免疫反应）。在此，我们研究了细胞因子诱导的杀伤（CIK）细胞与ALK和PD-1抑制剂联合对基因可变的NSCLC细胞系的抗肿瘤作用。
我们将三种基因不同的NSCLC细胞系NCI-H2228（EML4-ALK）、A549（KRAS突变）和HCC-78（ROS1重排）分别与纳武单抗（PD-1抑制剂）和克唑替尼（ALK抑制剂）进行共培养，分为有药和无药组。此外，我们使用流式细胞术以及RT-qPCR分析了CIK细胞表面多种免疫检查点表达的变异性、绝对死亡细胞的浓度、细胞内颗粒酶B的浓度。通过ELISA和蛋白质印迹法验证CIK细胞的活化情况。
我们的分析表明：（a）纳武单抗显著削弱了CIK细胞上PD-1的表面表达，而不影响其他免疫检查点或PD-1 mRNA表达；（b）这种联合策略对CD3 CD56 CIK细胞的细胞活力、IFN-γ产生和颗粒酶B的细胞内释放有有效反应，但仅在NCI-H2228细胞系中如此；（c）作为CIK细胞中T细胞活化标志物的Fas配体（FasL）的内在表达通过这种累加效应上调；（d）纳武单抗诱导CIK细胞CD4CD25亚群中Foxp3表达显著增加。综上所述，我们可以证明CIK细胞与克唑替尼和纳武单抗联合可通过FasL激活增强抗肿瘤免疫反应，导致IFN-γ和颗粒酶B增加，但仅在具有EML4-ALK重排的NCI-H2228细胞中如此。因此，我们假设CIK疗法可能是携带EML4-ALK重排的NSCLC患者的一种潜在替代疗法，此外，我们支持这样的观点，即联合疗法在根据患者个体进行优化时具有巨大潜力。
