肿瘤细胞治疗研究
英文原题：MICA/MICB-Mediated NKG2D Immune Escape in Cervical Cancer: Single-Cell Transcriptomic Mapping of Radionuclide Therapy Targets for Precision Radioimmunotherapy.
MICA/MICB-Mediated NKG2D Immune Escape in Cervical Cancer: Single-Cell Transcriptomic Mapping of Radionuclide Therapy Targets for Precision Radioimmunotherapy.
宫颈癌 scRNA-seq 图谱显示，分化簇 8（CD8）+ T 细胞显著减少（平均差：-0.12；95% CI：[-0.16, -0.08]；Cohen's d = 1.45），自然杀伤（NK）细胞亦显著减少（Cohen's d = 1.12），同时 CD25+ 调节性 T 细胞增加（+0.08；95% CI：[+0.05, +0.11]），从而确立了一个不利于 RNT 的免疫抑制性 TME。
宫颈癌具有深度免疫抑制的肿瘤微环境（TME），该微环境损害固有免疫和适应性抗肿瘤免疫，并且关键性地限制了新兴放射免疫治疗策略的疗效。NKG2D受体-配体轴——由应激诱导型配体MICA和MICB组成——构成了一个关键的固有免疫识别界面，其在肿瘤细胞表面的表达决定了NKG2D武装效应细胞的易感性，并进而决定了放射性标记NKG2D靶向探针用于精准放射性核素治疗（RNT）的可靶向性。然而，NKG2D配体失调的机制基础及其对宫颈癌放射性核素诊疗的意义仍不明确。本研究整合了单细胞RNA测序（scRNA-seq）和实验验证，全面绘制了宫颈癌发生过程中NKG2D轴免疫逃逸图谱，并阐明了其对精准RNT靶点选择和患者分层的转化意义。
scRNA-seq数据集（GSM1551311和GSM1551411）使用Seurat和Harmony进行处理，用于细胞类型注释、免疫景观表征和放射性核素靶点密度分析。在使用clustree包评估多个分辨率参数（0.4-1.2）以确保聚类分配稳定后，以0.8的分辨率进行Louvain聚类。基于肘部图分析，保留前20个主成分用于Uniform Manifold Approximation and Projection（UMAP）嵌入。Harmony整合使用默认参数（θ = 2和λ = 1），并在20次迭代中评估收敛性。使用DoubletFinder（v2.0.3）进行双细胞检测，估计双细胞率为4.0%；此外，排除了核糖体蛋白基因读数>40%的细胞。使用Local Inverse Simpson's Index、Adjusted Rand Index和轮廓系数指标验证批次校正质量。实时定量PCR和酶联免疫吸附试验（ELISA）定量了四种候选RNT相关基因——MICA、MICB（NKG2D配体；主要放射性核素靶向分子）、SUSD1（免疫抑制上调因子；潜在RNT耐药介质）和STAG3L1——在HeLa、SiHa和正常HCerEpiC细胞系中的表达。每种细胞系进行五个独立生物学重复，每个重复设三个技术重复，遵循Minimum Information for Publication of Quantitative Real-Time PCR Experiments（MIQE）指南。在所有参数分析之前，应用了Shapiro-Wilk正态性检验和Levene方差齐性检验。
宫颈癌 scRNA-seq 图谱显示，分化簇 8（CD8）+ T 细胞显著减少（平均差：-0.12；95% CI：[-0.16, -0.08]；Cohen's d = 1.45），自然杀伤（NK）细胞亦显著减少（Cohen's d = 1.12），同时 CD25+ 调节性 T 细胞增加（+0.08；95% CI：[+0.05, +0.11]），从而确立了一个不利于 RNT 的免疫抑制性 TME。与 RNT 应答型肿瘤类型——神经内分泌肿瘤和前列腺特异性膜抗原（PSMA）阳性前列腺癌——进行比较基准分析，证实宫颈癌表现出靶点表面密度降低、NKG2D 效应细胞群耗竭以及免疫抑制性亚群富集等特征，这些因素共同预示 RNT 疗效减弱。实验验证证实 MICA 显著下调（HeLa：相对表达量 0.44 ± 0.07，p < 0.001，n = 5）和 MICB 显著下调（HeLa：0.51 ± 0.09，p < 0.05），导致 MICA 蛋白分泌显著减少（HeLa 中为 124.3 ± 18.5 pg/mL，对照为 285.4 ± 31.2 pg/mL，p < 0.01）。同时，SUSD1 显著上调（HeLa：2.28 ± 0.25 倍；蛋白 3.42 ± 0.45 ng/mg，p < 0.001，n = 5）。mRNA-蛋白相关性较强，r = 0.78-0.92，p < 0.001；由每种细胞系五个独立生物学重复计算得出；所有测量的变异系数（CV）< 15%，验证了转录
BACKGROUND: Cervical cancer harbors a profoundly immunosuppressive tumor microenvironment (TME) that impairs innate and adaptive antitumor immunity and, critically, limits the efficacy of emerging radioimmunotherapy strategies. The NKG2D receptor-ligand axis-comprising the stress-inducible ligands MICA and MICB-constitutes a pivotal innate immune recognition interface whose surface expression on tumor cells determines susceptibility to NKG2D-armed effector cells and, by extension, dictates the targetability of radiolabeled NKG2D-directed probes for precision radionuclide therapy (RNT). Yet the mechanistic basis for NKG2D ligand dysregulation and its implications for radionuclide theranostics in cervical cancer remain poorly defined. This study integrated single-cell RNA sequencing (scRNA-seq) and experimental validation to comprehensively map the NKG2D-axis immune escape landscape in cervical carcinogenesis and to delineate its translational significance for precision RNT target selection and patient stratification. METHODS: scRNA-seq datasets (GSM1551311 and GSM1551411) were processed using Seurat and Harmony for cell-type annotation, immune landscape characterization, and radionuclide target density profiling. Louvain clustering was performed at a resolution of 0.8 after evaluating multiple resolution parameters (0.4-1.2) using the clustree package to ensure stable cluster assignments. The top 20 principal components were retained for Uniform Manifold Approximation and Projection (UMAP) embedding based on elbow plot analysis. Harmony integration used default parameters (θ = 2 and λ = 1) with convergence assessed over 20 iterations. Doublet detection was performed using DoubletFinder (v2.0.3) with an estimated doublet rate of 4.0%; additionally, cells with >40% ribosomal protein gene reads were excluded. Batch correction quality was validated using the Local Inverse Simpson's Index, Adjusted Rand Index, and silhouette coefficient metrics. Real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA) quantified expression of four candidate RNT-relevant genes-MICA, MICB (NKG2D ligands; primary radionuclide targeting molecules), SUSD1 (immunosuppressive upregulator; potential RNT resistance mediator), and STAG3L1-in HeLa, SiHa, and normal HCerEpiC cell lines. Five independent biological replicates were performed per cell line, each with three technical replicates, following Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Shapiro-Wilk normality testing and Levene's test for homogeneity of variance were applied prior to all parametric analyses. RESULTS: Cervical cancer scRNA-seq profiles revealed significantly depleted cluster of differentiation 8 (CD8)+ T cells (mean difference: -0.12; 95% CI: [-0.16, -0.08]; Cohen's d = 1.45) and natural killer (NK) cells (Cohen's d = 1.12), with increased CD25+ regulatory T cells (+0.08; 95% CI: [+0.05, +0.11]), establishing an RNT-unfavorable immunosuppressive TME. Comparative benchmarking against RNT-responsive tumor types, neuroendocrine tumors and prostate-specific membrane antigen (PSMA) positive prostate cancer, confirmed that cervical cancer exhibits a combination of reduced target surface density, depleted NKG2D-effector populations, and enriched immunosuppressive subsets collectively predictive of attenuated RNT efficacy. Experimental validation confirmed dramatic downregulation of MICA (HeLa: 0.44 ± 0.07 relative expression, p < 0.001, n = 5) and MICB (HeLa: 0.51 ± 0.09, p < 0.05), translating to markedly reduced MICA protein secretion (124.3 ± 18.5 pg/mL in HeLa versus 285.4 ± 31.2 pg/mL in controls, p < 0.01). Concurrently, SUSD1 was markedly upregulated (HeLa: 2.28 ± 0.25-fold; protein 3.42 ± 0.45 ng/mg, p < 0.001, n = 5). Strong mRNA-protein correlations, r = 0.78-0.92, p < 0.001; computed from five independent biological replicates per cell line; coefficient of variation (CV) < 15% for all measurements, validated transcr
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