Galectin-9 Emerges as a Key Driver of Immune Evasion in Cervical Cancer Progression

Cervical cancer continues to claim hundreds of thousands of lives each year, ranking as the fourth most common malignancy among women worldwide, and while vaccination against human papillomavirus has reshaped the long-term outlook for prevention, clinicians still lack precise molecular maps of how a healthy cervix slides step by step into malignancy. A new multi-omics study now offers one of the most detailed pictures yet of that transition, and in doing so it highlights a single protein, galectin-9, as a promising point of therapeutic attack. The research, published in Cancer Cell International, combines single-cell RNA sequencing, whole-genome bisulfite sequencing, and spatial transcriptomics to trace the immune landscape from normal cervical tissue through low-grade and high-grade squamous intraepithelial lesions to invasive squamous cell carcinoma and adenocarcinoma.

The study team, led by researchers at Zhejiang University and The Third Affiliated Hospital of Guangzhou Medical University, analyzed ten human cervical tissue samples spanning the full pathological continuum. Single-cell RNA sequencing allowed them to profile thousands of individual cells, resolving not only which cell types were present at each disease stage but also how their gene-expression programs shifted as lesions progressed. Whole-genome bisulfite sequencing added a crucial layer of epigenetic information, revealing how DNA methylation patterns change across cell types during carcinogenesis, while spatial transcriptomic data from a public cohort confirmed that the cellular relationships observed in dissociated single-cell data hold true within intact tissue architecture.

One of the most striking findings is that disease progression is accompanied by a marked increase in NK/T cell infiltration. As normal tissue advances through LSIL and HSIL toward invasive carcinoma, immune cells of the NK and T lineages crowd increasingly into the lesion environment. This might, at first glance, seem encouraging, since cytotoxic lymphocytes are the very cells capable of destroying tumor cells. Yet the infiltration coincides with upregulation of galectin-9, an immune checkpoint ligand encoded by the LGALS9 gene. Galectin-9 is known to bind Tim-3 on T cells, a interaction that dampens antitumor immunity and drives T-cell exhaustion. In other words, the tumor microenvironment appears to respond to escalating immune pressure by deploying an immunosuppressive ligand, a classic example of adaptive immune resistance.

The epigenetic data revealed something particularly interesting about how this deployment unfolds over time. The LGALS9 promoter underwent progressive demethylation beginning at the low-grade squamous intraepithelial lesion stage, indicating that the gene was being epigenetically primed for expression long before invasive cancer appeared. However, robust transcriptional induction of LGALS9 only became prominent at the high-grade lesion stage, coinciding with activation of interferon-gamma response programs. This temporal separation between demethylation and transcriptional activation suggests a two-step mechanism: early epigenetic poising followed by inflammatory triggering. The researchers found a remarkably tight correlation between interferon-gamma activity and LGALS9 expression at the sample level, with a Pearson correlation coefficient of 0.95, indicating that the very immune cells infiltrating the lesion may be inducing the ligand that ultimately silences them.

Beyond galectin-9, trajectory analysis of the single-cell data delineated the evolution of CD8-positive T cells across disease stages and identified TFCP2 as a transcriptional regulator whose activity is associated with patient prognosis. The study also documented enrichment of LAMP3-positive mature dendritic cells in tumor tissues compared with normal controls. These dendritic cells, which emerge along a maturation trajectory from conventional cDC1 and cDC2 subsets, carried a mixture of costimulatory molecules such as CD40 and CD80 and inhibitory checkpoint molecules including CD274, IDO1, and LGALS9 itself, suggesting that even the antigen-presenting arm of the immune response becomes entangled in the checkpoint machinery as cancer develops.

Multiplex immunohistochemistry provided protein-level confirmation of the story told by the sequencing data. Staining for exhausted CD8-positive T cells, marked by the co-expression of CD3, CD8, and Tim-3, alongside staining for the epithelial marker pan-cytokeratin and galectin-9, showed that galectin-9-positive epithelial cells increase in abundance as tissue progresses from normal cervix through LSIL and HSIL to cancer. The physical co-localization of Tim-3-expressing exhausted T cells with galectin-9-expressing epithelium within the same tissue sections strengthens the argument that this ligand-receptor pair represents a functional axis of immune evasion operating during precancerous progression, not merely a correlate of advanced disease.

Perhaps the most translational portion of the work came from animal experiments. The researchers established an ectopic subcutaneous syngeneic cervical cancer model in immunocompetent mice, an experimental system in which the immune system is fully intact and therefore capable of mounting genuine antitumor responses. Blocking galectin-9 in this model reduced tumor burden, demonstrating that the protein is not simply a passive biomarker but an active contributor to tumor growth. More strikingly, combining galectin-9 blockade with an agonist antibody against GITR, a costimulatory receptor on T cells, significantly enhanced the clonal expansion and cytotoxic activity of CD8-positive T cells. This combination strategy suggests that releasing one brake on the immune system while simultaneously pressing the accelerator may produce therapeutic effects greater than either intervention alone.

The findings arrive at a moment when immune checkpoint blockade has transformed the treatment of many cancers but has delivered comparatively modest results in cervical cancer. Understanding which checkpoint pathways are active at which stages of disease could allow clinicians to intervene earlier and more precisely. The observation that LGALS9 epigenetic poising begins at the LSIL stage is particularly provocative, since low-grade lesions are common, usually regress spontaneously, and are typically managed conservatively. If reliable markers of galectin-9 activation could be incorporated into screening algorithms, they might help distinguish the minority of low-grade lesions destined for progression from those that will resolve, sparing unnecessary procedures while directing attention to lesions that truly warrant close surveillance.

The study also illustrates the growing power of integrated multi-omics approaches in cancer biology. No single technology used here could have revealed the full sequence of events. Single-cell transcriptomics exposed the cellular composition and signaling programs of each lesion stage, but only DNA methylation profiling revealed that LGALS9 had been epigenetically prepared in advance of its expression, and only spatial transcriptomics could verify that the relevant cell populations occupy adjacent territories within intact tissue. Copy number variation inference, pseudotime trajectory modeling, and regulon analysis with tools such as pySCENIC and Monocle2 added further resolution, while methylation-based deconvolution using EpiSCORE extended the key NK/T cell infiltration trend across a larger cohort of twenty-one bulk tissue samples.

Caveats remain, as they do in any early-stage translational study. The human cohort comprised ten deeply profiled samples, and the syngeneic mouse model, while immunocompetent, does not fully recapitulate HPV-driven human cervical carcinogenesis. Clinical testing of galectin-9 blockade in cervical cancer patients would need to demonstrate safety and efficacy in the neoadjuvant, recurrent, or metastatic settings where immunotherapy is currently deployed. Nevertheless, by pinpointing a checkpoint ligand whose activation is detectable during precancerous progression and whose blockade shows antitumor efficacy in vivo, the study provides both a mechanistic framework for understanding immune evasion in cervical carcinogenesis and a concrete, testable therapeutic hypothesis. For a disease that remains a leading cause of cancer death among women globally, that combination of mechanistic insight and actionable target represents a meaningful step forward.

Subject of Research: Multi-omics analysis of immune evasion during cervical carcinogenesis and galectin-9 as a candidate immunotherapeutic target

Article Title: Multi-omics analysis of cervical carcinogenesis reveals galectin-9 driven immune evasion as a candidate immunotherapeutic target

Article References: Multi-omics analysis of cervical carcinogenesis reveals galectin-9 driven immune evasion as a candidate immunotherapeutic target. (n.d.). https://doi.org/10.1186/s12935-026-04458-1

Image Credits: AI Generated

DOI: 10.1186/s12935-026-04458-1

Keywords: cervical cancer, galectin-9, LGALS9, single-cell RNA sequencing, spatial transcriptomics, DNA methylation, CD8-positive T cells, immune evasion, immunotherapy, tumor microenvironment, interferon-gamma, cervical carcinogenesis