Abstract
Cervical cancer (CC) remains the fourth most common malignancy among women worldwide. While single-cell studies have revealed heterogeneity in the tumor microenvironment (TME) of invasive CC, the dynamic changes and regulatory mechanisms that drive precancerous progression remain poorly characterized. Using single-cell RNA sequencing of ten human cervical tissues that span the pathological continuum from normal cervix through low-grade and high-grade squamous intraepithelial lesions (LSIL and HSIL) to squamous cell carcinoma (SCC) and cervical adenocarcinoma (AD), complemented by whole-genome bisulfite sequencing (WGBS) and spatial transcriptomic validation, we found that disease progression is accompanied by a marked increase in NK/T cell infiltration and by upregulation of the immune checkpoint ligand Galectin-9 (encoded by LGALS9). LGALS9 underwent progressive promoter demethylation from the LSIL stage onward, whereas its transcriptional induction became prominent at HSIL in association with interferon-gamma response activation. Trajectory analysis delineated the evolution of CD8⁺ T cells and identified TFCP2 as a transcriptional regulator associated with patient prognosis. Tumor tissues were enriched in LAMP3⁺ mature dendritic cells (mDCs) compared to normal controls. In vivo, in an ectopic subcutaneous syngeneic cervical cancer model established in immunocompetent mice, Galectin-9 blockade reduced tumor burden, and its combination with an anti-GITR agonist significantly enhanced CD8⁺ T cell clonal expansion and cytotoxicity. These findings provide deeper insight into CC initiation and progression and nominate Galectin-9 as a candidate immunotherapeutic target: its activation is detectable during precancerous progression, and its blockade shows antitumor efficacy in a syngeneic cervical cancer model.