Integration of Flow Cytometry and Single-Cell RNA Sequencing Analysis to Explore the Fibroblast Subpopulations in Keloid that Correlate with Recurrence.

Abstract

Objective: Fibroblasts (FBs) are the cytological basis of keloid (KD) formation. This study aimed to identify the key pathogenic target cell subpopulation involved in KD recurrence. Approach: Single-cell RNA sequencing data were retrieved from public databases, revealing distinct gene expression patterns in FB subpopulations. Flow cytometry (FCM) was used to identify the surface molecular phenotypes of FBs that affect KD recurrence. Simultaneously, logistic regression analysis was performed to assess the predictive value of changes in FB subpopulation percentages for clinical KD recurrence. Results: The percentage of keloid fibroblasts was significantly greater than that in normal tissues. Through further clustering analysis of the FB population, we obtained four subpopulations, FB1-FB4, in which the percentages of FB1 subpopulation were increased, and functional enrichment analysis suggested that the FB1 subpopulation may play a greater role in extracellular matrix collagen oversynthesis in KD. In addition, the gene expression of CD26 (DPP4), CD117 (c-KIT), and CD34 in the FB1 subpopulation was significantly higher than that in FB2-4 subpopulations. Moreover, the percentage of CD26+/CD117+/CD34+ cell subpopulations in the FCM data of patients with KD recurrence was significantly increased. Regression analysis confirmed that the CD26+/CD117+/CD34+ FB subpopulation was a risk factor for relapse. Innovation: We demonstrated that the molecular phenotypic and functional heterogeneity of FBs influences KD recurrence. Conclusion: We identified key pathogenic FB subpopulations that may affect KD development, which can be used as potential markers to predict recurrence and provide potential target cell populations for future clinical treatment.