Visium expression matrices

Biomarker-assisted screening may improve diagnostic accuracy, but biomarkers predicting progression of low-grade squamous intraepithelial lesions (LSIL) remain limited.</p><p dir="ltr"><b>Methods</b>: We integrated single-cell RNA sequencing and spatial transcriptomics to identify prognostic biomarkers associated with LSIL outcomes, followed by validation using improved <i>in situ</i> sequencing (IISS).</p><p dir="ltr"><b>Results</b>: Analysis of 341,978 single cells identified eight major cell populations and twelve epithelial subclusters, including a previously unrecognized subcluster, epi8.

Although mainly derived from normal cervix, epi8 exhibited cancer-associated features and shared molecular characteristics with the prognostic biomarkers <i>FAM19A4</i> and <i>miR124-2</i>. <i>In situ</i> sequencing showed that epi8 localized adjacent to HPV-infected basal cells, with stage-dependent spatial changes during disease progression. Retrospective analysis further demonstrated that epi8-associated markers stratified LSIL into progression, persistence, and regression groups, with <i>CRYAB</i>^{<em>high</em>}<i>/GAS5</i>^{<em>high</em>} associated with progression and <i>RPLP1</i>^{<em>high</em>}<i>/SNHG5</i>^{<em>high</em>} associated with regression.</p><p dir="ltr"><b>Conclusions</b> We identified a previously unrecognized epithelial subcluster associated with early cervical carcinogenesis and demonstrated that its molecular markers enable risk stratification of LSIL, providing candidate biomarkers for precision triage in cervical cancer screening.</p>