Integrative Single-Cell and Spatial Transcriptomic Analysis Reveals MAIT Cell Dysfunction in Relapsed HCC

Hepatocellular carcinoma (HCC) frequently recurs after curative treatment, and the tumor immune microenvironment plays an important role in disease progression. However, the role of mucosal-associated invariant T (MAIT) cells in relapsed HCC remains poorly understood. This study aimed to characterize transcriptional and spatial features of MAIT cells in relapsed HCC and their association with malignant hepatocyte phenotypes.

Tumor samples from primary (n = 3) and relapsed (n = 2) HCC patients were analyzed using paired single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics. scRNA-seq data (49,229 cells) were processed using Seurat with standard quality-control thresholds, followed by Harmony batch correction and unsupervised clustering. Malignant hepatocytes were identified by copy-number variation inference. Spatial transcriptomic data from 35 regions of interest were normalized and deconvolved using scRNA-seq-derived reference profiles. Independent validation was performed using a public HCC scRNA-seq dataset.^1.

Integrated analyses revealed distinct tumor microenvironmental features in relapsed HCC. Relapsed tumors showed increased representation of malignant hepatocytes with elevated cancer stemness-related transcriptional signatures compared with primary tumors (1.18-fold increase, p < 0.0001), which was spatially supported by enrichment in tumor regions (1.10-fold increase, p ≤ 0.05). Within the T/NK compartment, MAIT cells were significantly enriched in relapsed tumor regions (2.71-fold increase, p ≤ 0.05). Transcriptomic profiling identified distinct MAIT cell states between primary and relapsed HCC, with relapsed MAIT cells displaying dysfunctional phenotype. Cell-cell interaction analysis suggested enhanced ligand-receptor interactions between MAIT cells and malignant hepatocytes in relapsed tumors. In the TCGA LIHC cohort, high relapsed MAIT cell signature scores were associated with poorer overall survival (HR = 1.52, p ≤ 0.05).

Relapsed HCC is characterized by enhanced malignant hepatocyte stemness and altered MAIT cell states within the tumor microenvironment. These findings suggest an association between MAIT cell dysregulation and relapse-specific tumor biology, warranting further functional investigation.