MICA/MICB-Mediated NKG2D Immune Escape in Cervical Cancer: Single-Cell Transcriptomic Mapping of Radionuclide Therapy...

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.

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.

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 transcriptomic profiling as a reliable proxy for theranostic target protein density estimation.

This integrative study reveals that MICA/MICB downregulation and SUSD1 upregulation converge to suppress NKG2D-mediated antitumor immunity in cervical cancer, creating an immune-cold TME that limits current immunotherapy and radionuclide targeting efficacy. The NKG2D ligand expression landscape mapped here delineates a precision RNT strategy: scRNA-seq-guided patient stratification, radiolabeled anti-MICA/MICB nanobody theranostic imaging to confirm surface target density, and combination radioimmunotherapy integrating MICA/MICB re-expression induction with targeted radionuclide delivery to selectively irradiate the NKG2D-ligand-negative tumor cell population.