Histopathology-guided spatial transcriptomic analysis identifies an ECM-associated stromal pattern in lacrimal gland...

Lacrimal gland adenoid cystic carcinoma (LGACC) is a rare but locally aggressive malignancy characterized by perineural invasion, recurrence, and limited systemic treatment options. Although extracellular matrix (ECM) remodeling is increasingly recognized as an important regulator of tumor progression, its spatial organization and functional relevance in LGACC remain incompletely understood.

A publicly available LGACC spatial transcriptomic tissue section was reanalyzed using histopathology-guided annotation to define a highly cellular Tumor Core and an adjacent stromal region, termed the Stroma Boundary. Regional differential expression and functional enrichment analyses were performed, together with module scoring, a sensitivity analysis within a shared transcriptomic cluster spanning both histopathologically defined regions, and CellChat-based ligand-receptor inference. The spatial findings were further contextualized using a public bulk RNA-sequencing cohort and an in-house quantitative proteomic cohort comparing conventional LGACC with LGACC showing high-grade transformation (LGACC-HGT). The effects of type I collagen were evaluated in SACC-LM cells using morphological assessment, CCK-8, wound-healing, Transwell migration, and Western blotting assays.

Within the analyzed spatial section, Stroma Boundary spots showed higher expression of ECM-related genes, including COL1A1, COL1A2, SPARC, and DCN, together with enrichment of collagen fibril organization and extracellular matrix organization. A secondary analysis restricted to a shared transcriptomic cluster spanning both histopathologically defined regions showed that ECM-associated differences remained detectable within a more similar transcriptomic background. CellChat analysis nominated collagen-associated interactions involving CD44 as candidate components of region-to-region communication. Bulk RNA-sequencing provided contextual support for the tumor-associated transcriptional program but incompletely captured the localized stromal pattern. Proteomic profiling demonstrated increased abundance of multiple collagen- and matrix-associated proteins in LGACC-HGT, accompanied by enrichment of ECM-receptor interaction and focal adhesion-related pathways. In vitro, type I collagen induced a more dispersed and spread cellular morphology and enhanced wound closure and Transwell migration without significantly altering short-term cell viability. Collagen exposure was also associated with increased CD44 protein expression. CD44 knockdown attenuated the COL1-induced increase in wound closure, suggesting a functional contribution of CD44 to this response.

Histopathology-guided spatial analysis identified an ECM-enriched regional transcriptional pattern in the stromal compartment of the analyzed LGACC section. Complementary proteomic and functional findings suggest that collagen-associated remodeling may contribute to the migratory phenotype of ACC cells. CD44 represents a biologically plausible candidate associated with the collagen response, although its mechanistic role requires further validation.