"Eye-Conic" Spatial Transcriptomics Reveals the Layer-Specific Molecular Alterations in Corneas of Patients With...

Due to the structural complexity of the human cornea as well as the characteristic histopathological changes occurring in keratoconus (KTCN) cornea, there is a need to identify the associated cellular and molecular layer-specific alterations in the process of KTCN cone formation.

The spatial transcriptomics (ST) approach was applied for KTCN and control corneas to investigate the layer-specific alterations. The data, organized into spatial clusters corresponding to corneal layers, were subjected to bioinformatic analyses, including pathways enrichment, spots deconvolution, and cell-cell communication network inference. Additionally, the outputs derived from our previous bulk RNA-seq experiment and immunofluorescence staining of corneal cryosections were evaluated.

The findings encompassing (i) deregulation of cell cycle, and peripheral accumulation of cell-cell junctions, pointing to a disrupted process of corneal epithelium (CE) renewal; (ii) upregulated extracellular matrix (ECM)-related pathways and enriched hallmark of epithelial-mesenchymal transition, affecting the stromal wound healing; (iii) activation of "Translation" and "Ribosome" pathways in the endothelium pointing to disrupted metabolic activity; and (iv) a unique cellular composition of KTCN cornea, with substantial stromal abnormalities. Furthermore, the downregulated expression of MT2A and SAA1 genes, linked to cellular responses to stress and/or oxidative stress, was revealed across all KTCN corneal layers, corresponding to the identified underrepresentation of specific cell types. By comparing less- and more advanced KTCN phenotypes, the characteristic intercellular communication patterns were recognized.

Findings regarding the corneal layers and the intercellular communication patterns in KTCN corneas allowed for further understanding of the cone formation mechanism.