Background: Basal cell carcinoma (BCC) represents the most common human malignancy, with aggressive histopathological subtypes posing significant clinical challenges due to their potential for local tissue destruction and rare metastatic capability. While histopathological classification distinguishes low- from high-risk variants, molecular characterization of these subtypes remains limited.

Methods: We examined estrogen receptor alpha (ESR1) expression patterns across BCC subtypes using spatial analysis techniques. Quantitative image analysis was performed to assess ESR1 distribution in both malignant keratinocytes and adjacent stromal fibroblasts, with particular attention to tumor nest architecture and stromal relationships. Cyclic immunofluorescence and single-cell RNA sequencing (scRNAseq) were employed to validate spatial interactions and characterize specific tumor–fibroblast communication pathways.

Results: High-risk BCC subtypes demonstrated significantly elevated focal ESR1 expression compared to low-risk variants. Notably, infiltrative tumor regions exhibited prominent ESR1 positivity at tumor lobe peripheries. Quantitative spatial analysis revealed a positive correlation between tumor nest size and ESR1 expression in the adjacent stroma, suggesting coordinated tumor–stromal signaling. Cyclic immunofluorescence confirmed these spatial interactions, while scRNAseq analysis revealed specific molecular communication pathways between ESR1-positive tumor cells and stromal fibroblasts.

Conclusions: Our findings identify ESR1 as a novel molecular marker distinguishing aggressive BCC subtypes and reveal previously unrecognized estrogen receptor signaling in BCC pathogenesis. The spatial relationship between tumor architecture and stromal ESR1 expression suggests that estrogen signaling may contribute to the invasive behavior characteristic of high-risk BCC variants, offering potential therapeutic targets for aggressive disease.