Background and Aims: CD4 ⁺ regulatory T cells (Tregs) are pivotal in HCC progression. However, systemic depletion of all Tregs risks autoimmunity. Existing subgroup classifications highlight Tregs’ phenotypic and functional heterogeneity but lack coherent consensus. This study aimed to classify Treg subtypes using single-cell CITE-seq, integrating data from tumor, non-tumor, and blood samples in HCC patients. We also validated the clinical relevance and prognostic value of this classification. Approach and Results: CITE-seq analysis was performed on 51,067 CD4 ⁺ T cells from 8 HCC patients. Validation involved 96 HCC patients and 53 healthy donors using flow cytometry, functional assays, and clinical data. Trajectory and TCR analyses identified a peripheral Foxp3 ^high Treg subset that preferentially migrates to tumor sites, acquiring a terminally differentiated, activated phenotype. These tumor-infiltrating Foxp3 ^high Tregs exhibit elevated LAYN and TRM signatures and further increase their Foxp3 expression and immunosuppressive functions in response to pro-inflammatory cytokines in tumor tissue. The CCL5/CCR5 axis is crucial for recruiting Foxp3 ^high Tregs from peripheral blood into the tumor microenvironment. A strong correlation was observed between the percentage of Foxp3 ^high Tregs in peripheral blood and their counterparts in tumor regions, suggesting their potential as peripheral biomarkers. Notably, a peripheral Foxp3 ^high Tregs/CD4 ⁺ T cells percentage >3.5% predicted overall survival and early recurrence, with AUROCs exceeding 0.75. Conclusions: Peripheral Foxp3 ^high Tregs migrate to tumor sites via the CCR5–CCL5 axis and mature in response to pro-inflammatory cytokines. The proportion of these Tregs in peripheral blood correlates with their presence in tumors, making them a potential biomarker for predicting HCC outcomes.

Oroxylin A (Oro-A), the main bioactive flavonoid extracted from Scutellaria radix, has been reported to inhibit migration in various human cancer cell models. In this study, we further explored the anti-migration effects of Oro-A on oral squamous cell carcinoma (OSCC) cells and investigated the underlying mechanisms. A 24-h (short-term) exposure of OSCC cells to non-cytotoxic concentrations (5–20 μM) of Oro-A significantly suppressed cell migration according to a wound-healing assay. Furthermore, a 30-day exposure (long-term) to Oro-A (20 μM), which did not exhibit a cytotoxic effect on OSCC cells, significantly suppressed cell migration more than short-term Oro-A exposure. To uncover the molecular mechanisms underlying the inhibitory effect of long-term Oro-A exposure on OSCC migration, a cDNA microarray and the Ingenuity software were used. Overall, 112 upregulated and 356 downregulated genes were identified in long-term Oro-A-exposed cells compared with untreated OSCC cells. Among them, 75 genes were reported to be associated with cancer cell migration. Consistent with the cDNA microarray results, we found that the expression levels of several cell migration-related genes, such as LCN2, ID-1, MDK, S100A9 and CCL2, were significantly decreased in long-term Oro-A-exposed OSCC cells using a quantitative real-time polymerase chain reaction (Q-PCR) assay. The Western blotting and enzyme-linked immunosorbent assay (ELISA) results also demonstrated that CCL2 expression at the mRNA and protein levels was significantly decreased in long-term Oro-A-exposed OSCC cells compared with untreated OSCC cells. Moreover, the expression levels of downstream CCL2 targets, including p-ERK1/2, NFκB, MMP2, and MMP9, were also decreased in long-term Oro-A-exposed OSCC cells. Further, Oro-A treatment suppressed in vivo metastasis. These results suggest that long-term Oro-A treatment inhibits metastasis via CCL2 signaling in OSCC cells.