“Eye-Conic” Spatial Transcriptomics Reveals the Layer-Specific Molecular Alterations in Corneas of Patients With Keratoconus

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