Targeting CircZKSCAN1 suppresses M2 macrophage polarization and activates anti-tumor immunity in head and neck squamous cell carcinoma

Objective Malignant progression of head and neck squamous cell carcinoma (HNSCC) is closely associated with its immunosuppressive microenvironment. This study aimed to determine, for the first time in HNSCC, the regulatory role of circZKSCAN1 in the tumor immune microenvironment in an in vivo SCC4 xenograft model and to explore how it influences macrophage polarization. Methods SCC4 cells with or without circZKSCAN1 knockdown were implanted into BALB/c nude mice (three groups, n = 10 each): Model group, Negative control siRNA (si-NC) group, and CircZKSCAN1 siRNA (si-ZKSCAN1) group. Tumor growth and circZKSCAN1 expression were assessed by caliper measurements and quantitative real-time PCR, while intratumoral CD4 + /CD8 + T cells, macrophage polarization markers, and cytokine profiles were evaluated by flow cytometry, immunohistochemistry, and Western blotting. Results No significant differences were observed in any measured parameter between the Model group and the si-NC group ( P > 0.05). Compared with the above two groups, the si-ZKSCAN1 group exhibited significantly decreased circZKSCAN1 expression levels, significantly reduced xenograft tumor volume and weight, significantly decreased proportions of CD4 + and CD8 + T cells, a significantly increased proportion of inducible nitric oxide synthase-positive cells (an M1 macrophage marker), significantly decreased proportions of F4/80-, CD206-, and transforming growth factor-β-positive cells (M2 macrophage markers), significantly increased protein expression of the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α, and significantly decreased protein expression of the anti-inflammatory cytokine interleukin-10 and the M2-associated protein arginase-1 ( P < 0.05). Conclusion In a BALB/c nude mouse SCC4 xenograft model, circZKSCAN1 silencing inhibits tumor growth and is associated with an M2-to-M1 macrophage shift and remodeling of the local immune milieu. These in vivo findings suggest circZKSCAN1 as a potential immunotherapeutic target in HNSCC, which now requires validation in additional models and human tissues.