Single-Cell RNA-Seq Reveals a Glycolysis-Biased EMT State in Keloid Keratinocytes That Amplifies the Pro-Inflammatory and Fibrotic Microenvironment.

BACKGROUND

Keloids are pathological fibroproliferative skin disorders resulting from abnormal wound healing, characterized by excessive extracellular matrix (ECM) deposition and chronic local inflammation. While dermal fibroblasts are the primary effector cells in this process, the role of keloid keratinocytes (KKs) and their metabolic alterations in sustaining the pro-inflammatory and fibrotic microenvironment remains poorly understood.

METHODS

We performed single-cell RNA sequencing (scRNA-seq) on 46,690 cells from keloid and normal skin tissues. Cellular metabolic activities were assessed using scMetabolism and scFEA, and transcriptional regulatory networks were analyzed via pySCENIC. In vitro experiments utilizing glycolysis inhibitors (2-DG and Fasentin) were conducted to validate the relationship between metabolic reprogramming and epithelial-mesenchymal transition (EMT).

RESULTS

scRNA-seq analysis identified a distinct subpopulation of basal keratinocytes (Basal1/2) exhibiting an arrested differentiation trajectory and elevated glycolytic activity. This metabolic shift is functionally coupled with an EMT-prone state and is regulated by transcription factors including REL and ESRRA. Pseudotime analysis revealed that these KKs transition into a terminal state characterized by high expression of the chemokine CCL2 and the stress-response gene ATF3. Cell-cell communication analysis indicated that these glycolytically active KKs maintain intensive crosstalk with a specific pro-inflammatory fibroblast subset (Fibroblasts3) via the ligand-receptor pair SEMA3C-EPHA2 and IGFBP3-ADGRG1 signaling pathways. In vitro experiments confirmed that pharmacological inhibition of glycolysis with 2-DG or Fasentin significantly attenuated the EMT-related features in KKs.

CONCLUSION

Collectively, glycolysis-driven EMT in KKs actively contributes to the pro-inflammatory and fibrotic signaling in keloids, suggesting that targeting this metabolic reprogramming could be a potential strategy for therapeutic intervention.