基于单细胞技术的胰腺癌免疫微环境相关铜死亡相关 lncRNAs 的开发和验证。

BACKGROUND: Cuproptosis, a novel mode of cell death associated with the tricarboxylic acid (TCA) cycle, is relevant to the development of cancer. However, the impact of single-cell-based Cuproptosis-associated lncRNAs on the Tumor immune microenvironment (TIME) of Pancreatic adenocarcinoma (PAAD) and its potential value for individualized immunotherapy has not been clarified. METHODS: 14 immune-related CRGs were screened by exploring the interaction between differentially expressed Immune-Related Genes (IRGs) and Cuproptosis-Related Genes (CRGs) in PAAD. Next, the expression amount and expression distribution of CRGs in single-cell samples were analyzed by focusing on 7-CRGs with significant expressions. On the one hand, MAP2K2, SOD1, and VEGFA, which were significantly differentially expressed between PAAD sites and normal tissues adjacent to them, were subjected to immunohistochemical validation and immune landscape analysis. On the other hand, from these 7-CRGs, prognostic signatures of lncRNAs were established by co-expression and LASSO-COX regression analysis, and their prognostic value and immune relevance were assessed. In addition, this study not only validated the hub CRGs and the lncRNAs constituting the signature in a PAAD animal model treated with immunotherapy-based combination therapy using immunohistochemistry and qRT-PCR but also explored the potential value of the combination of targeted, chemotherapy and immunotherapy. RESULTS: Based on the screening of 7-CRGs significantly expressed in a PAAD single-cell cohort and their co-expressed Cuproptosis-Related lncRNAs (CRIs), this study constructed a prognostic signature of 4-CRIs named CIR-score. A Nomogram integrating the CIR-score and clinical risk factors was constructed on this basis to predict the individualized survival of patients. Moreover, high and low-risk groups classified according to the median of signatures exhibited significant differences in clinical prognosis, immune landscape, bioenrichment, tumor burden, and drug sensitivity. And the immunohistochemical and qRT-PCR results of different mouse PAAD treatment strategies were consistent with the trend of inter-group variability in drug sensitivity of hub CRGs and CIR-score. The combination of immunotherapy, targeted therapy, and chemotherapy exhibited a better tumor suppression effect. CONCLUSION: CIR-score, as a Cuproptosis-related TIME-specific prognostic signature based on PAAD single cells, not only predicts the prognosis and immune landscape of PAAD patients but also provides a new strategy for individualized immunotherapy-based combination therapy.

背景：铜死亡是一种与三羧酸（TCA）循环相关的新型细胞死亡模式，与癌症的发展有关。然而，基于单细胞的铜死亡相关长链非编码 RNA（lncRNAs）对胰腺导管腺癌（PAAD）肿瘤免疫微环境（TIME）的影响及其对个体化免疫治疗的潜在价值尚未阐明。

方法：通过探讨差异表达的免疫相关基因（IRGs）与 PAAD 中铜死亡相关基因（CRGs）之间的相互作用，筛选出 14 个免疫相关 CRGs。接下来，通过分析具有显著表达的 7 个 CRGs，重点关注单个细胞样本中 CRGs 的表达量和表达分布。一方面，对在 PAAD 部位和邻近正常组织之间差异表达显著的 MAP2K2、SOD1 和 VEGFA 进行免疫组织化学验证和免疫景观分析。另一方面，从这 7 个 CRGs 中，通过共表达和 LASSO-COX 回归分析建立 lncRNAs 的预后特征，并评估其预后价值和免疫相关性。此外，本研究不仅通过免疫组织化学和 qRT-PCR 验证了在接受基于免疫治疗的联合治疗的 PAAD 动物模型中治疗的标志性 CRGs 和构成特征的 lncRNAs，还探讨了靶向、化疗和免疫治疗相结合的潜在价值。

结果：基于在 PAAD 单细胞队列中显著表达的 7 个 CRGs 及其共表达的铜死亡相关 lncRNAs（CRIs）的筛选，本研究构建了一个命名为 CIR-score 的 4 个 CRIs 的预后特征。在此基础上，构建了一个包含 CIR-score 和临床危险因素的列线图，以预测患者的个体化生存。此外，根据特征中位数对高风险和低风险组进行分类，在临床预后、免疫景观、生物富集、肿瘤负担和药物敏感性方面存在显著差异。并且不同的小鼠 PAAD 治疗策略的免疫组织化学和 qRT-PCR 结果与特征中标志性 CRGs 和 CIR-score 的组间变异性趋势一致。免疫治疗、靶向治疗和化疗的联合治疗表现出更好的肿瘤抑制效果。

结论：基于 PAAD 单细胞的 CIR-score 是一种与铜死亡相关的 TIME 特异性预后特征，不仅可以预测 PAAD 患者的预后和免疫景观，还为基于个体化免疫治疗的联合治疗提供了一种新策略。