PD-L1 and IFN-γ modulate Non-Small Cell Lung Cancer (NSCLC) Cell Plasticity responsible for ICI-Resistance

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Although immune checkpoint inhibitors (ICIs) have demonstrated outstanding clinical efficacy in the treatment of NSCLC patients, ICIs-based therapy has also been associated with a paradoxical acceleration of tumor growth defined as hyperprogressive disease (HPD). In this paper we evaluated distinct plasticity traits in a model of HPD-NSCLC, with the aim of clarifying the mechanisms contributing to ICI resistance that involve IFN-γ and PD-L1. For this purpose, primary cell cultures were established from two stage IV NSCLC samples obtained from a single patient: one, prior to ICI initiation (NSCLC-B, baseline) and the other at the time of radiological evidence of hyperprogression under ICI treatment (NSCLC-H, hyperprogression). Compared to NSCLC-B cells, NSCLC-H cells exhibited a more aggressive in vivo and in vitro behavior, higher MAPK activation and owned the ability to develop tumoroids. The 3500 differentially expressed transcripts, according to whole transcriptome analysis, well described the profound plastic evolution of cells from NSCLC-B to NSCLC-H culture, as well as the increase of CD44 in the NSCLC-H cell line model, in which transcripts of CD44 isoforms lacking the variant domain were abundant. Transcripts of genes involved in the cellular response to IFN-γ were down-modulated in NSCLC-H compared to NSCLC-B, together with CD274. Conversely, an up-regulation of genes related to inflammatory response, including IL1-𝛽 and IFNGR1, was found. In vitro response to IFN- γ was compromised in both NSCLC-B and NSCLC-H cells, since IFN- γ failed to exert its antiproliferative effect on these cells and to effectively induce PD-L1 expression in 2D-growth assays. Nevertheless, the cytokine induced the activation of both type I and type II IFN-pathway mediators. In addition, treatment with IFN-γ induced NSCLC-H traits in NSCLC-B cells, promoting a striking increase in the number of NSCLC-B 3D-soft agar colonies. Low IFN-γ doses or modulation of PD-L1 contributed to the evolution of NSCLC-B towards NSCLC-H phenotype through the augment of CD44 on cell membrane, cell morphological changes and increased cell growth or sphere formation ability. In conclusion, we report a modulation of plasticity by PD-L1 modulation and IFN-γ in a NSCLC cell culture established from a treatment-naïve patient, who developed HPD under ICI therapy, suggesting an association between NSCLC plasticity and these main actors involved in ICI activity. Primary cell cultures from two stage IV NSCLC samples from same patient prior and after ICI treatment

非小细胞肺癌（Non-small cell lung cancer, NSCLC）是全球范围内癌症相关死亡的首要病因。尽管免疫检查点抑制剂（immune checkpoint inhibitors, ICIs）在非小细胞肺癌患者的治疗中展现出卓越的临床疗效，但基于免疫检查点抑制剂的治疗却可能引发一种矛盾的肿瘤生长加速现象，即超进展性疾病（hyperprogressive disease, HPD）。本研究针对非小细胞肺癌超进展模型中的不同可塑性特征展开评估，旨在阐明涉及干扰素γ（IFN-γ）与程序性死亡受体配体1（PD-L1）的免疫检查点抑制剂耐药机制。为此，研究人员从同一名患者的2份IV期非小细胞肺癌样本中建立原代细胞培养体系：一份采集于免疫检查点抑制剂治疗启动前（命名为NSCLC-B，基线组），另一份采集于免疫检查点抑制剂治疗期间经影像学证实出现超进展时（命名为NSCLC-H，超进展组）。与NSCLC-B细胞相比，NSCLC-H细胞在体内外均表现出更强的侵袭性，丝裂原活化蛋白激酶（MAPK）激活水平更高，且具备构建肿瘤类器官的能力。全转录组分析鉴定出的3500个差异表达转录本，清晰揭示了从NSCLC-B到NSCLC-H细胞培养体系的深刻可塑性演化，同时也体现出NSCLC-H细胞系中CD44的表达上调——该细胞系中缺乏变异结构域的CD44转录本丰度显著升高。相较于NSCLC-B细胞，NSCLC-H细胞中参与干扰素γ细胞应答的基因转录本以及CD274均被下调。与之相反，与炎症应答相关的基因（包括白细胞介素1β（IL-1β）与干扰素γ受体1（IFNGR1））则呈现上调表达。体外实验显示，NSCLC-B与NSCLC-H细胞均对干扰素γ应答受损：干扰素γ无法对这两种细胞发挥抗增殖作用，也无法在二维培养实验中有效诱导PD-L1的表达。不过，该细胞因子仍可激活I型与II型干扰素通路的介导分子。此外，干扰素γ处理可在NSCLC-B细胞中诱导出NSCLC-H的表型特征，显著增加NSCLC-B细胞的软琼脂三维集落数量。低剂量干扰素γ或PD-L1调控可通过上调细胞膜CD44表达、改变细胞形态以及增强细胞增殖或成球能力，推动NSCLC-B细胞向NSCLC-H表型转化。综上，本研究报道了在一名接受免疫检查点抑制剂治疗后出现超进展的初治非小细胞肺癌患者的原代细胞培养体系中，PD-L1调控与干扰素γ可对细胞可塑性产生调控作用，提示非小细胞肺癌的细胞可塑性与免疫检查点抑制剂活性相关的两大核心因子存在关联。本研究的细胞培养体系来源于同一名患者在免疫检查点抑制剂治疗前后获取的2份IV期非小细胞肺癌样本。