MYOFIBROBLASTS IN SSC ARE A HETEROGENEOUS POPULATION WITH EPIGENETICALLY STABLE PHENOTYPE IN VITRO.

Caballero-Ruiz, B.; Ross, R.; Corinaldesi, C.; Philippeos, C.; Watt, F.; Riobo-Del Galdo, N.; Del Galdo, F.

MYOFIBROBLASTS IN SSC ARE A HETEROGENEOUS POPULATION WITH EPIGENETICALLY STABLE PHENOTYPE IN VITRO.

All Authors

Caballero-Ruiz, B.

Ross, R.

Corinaldesi, C.

Philippeos, C.

Watt, F.

Riobo-Del Galdo, N.

Del Galdo, F.

LTHT Author

Ross, Rebecca
Del Galdo, Francesco

LTHT Department

NIHR Leeds Biomedical Research Centre
Rheumatology

Publication Date

2024

Item Type

Conference Abstract

Abstract

Introduction: Most of the cellular and molecular biology knowledge acquired on the pro-inflammatory and profibrotic activation of dermal fibroblasts in Systemic Sclerosis (SSc) has derived from analysis of cellular function and RNA/protein expression of dermal fibroblasts sub-cultured from skin biopsies. The heterogeneity of the findings across fibroblast lines and over passages is a well acknowledged phenomenon in the field. Recent single cell Sequencing of SSc skin biopsies has offered a potential explanation to the heterogeneity by confirming the existence of distinct dermal fibroblasts "endotypes" with variable prevalence of specific population across patients and over time. Here we established stable cultures form single cell clones of SSc fibroblasts to characterize their function and inform next generation of cellular and molecular studies on these cells. Material(s) and Method(s): Dermal fibroblasts from skin biopsies of Healthy (N=3) or Diffuse cutaneous SSc (N=3) patients were isolated, immortalized, and serially diluted to create single cell clone cell lines. RT-PCR and RNAseq was used to study differential transcriptomes and changes in protein levels (western blotting). In vitro phenotypic assays (collagen contraction, de-epidermised dermis (DED), proliferation assay) and endothelial co-cultured tubulogenesis assays. Result(s): Firstly, we observed a highly heterogenous rate of proliferation across SSc. We selected 7 SSc clones with different proliferation rates and 3 "reference" HC clones for future analysis. Transcriptome analysis by RNAseq revealed that SSc clones clustered separately from healthy controls. The SSc cluster was driven by "classical" profibrotic genes including SFRP2, ACTA2, CNN2 confirming that the cloning procedure did not alter the biological signature of SSc fibroblasts. SSc clones showed overall increased protein levels of CCN2 and SMA as well as increased gel contraction ability compared to healthy controls. Similarly, at group level there was an overall decreased ability to sustain endothelial cell tubulogenesis in organotypic co-culture models. Nevertheless, SSc clones showed important differences in their transcriptome including OX40L, IL-6, SFRP2/4, COMP, THBS1 and LRG5. Interestingly the clones expressing high LRG5 expression were also the ones with smallest proliferation rate, supporting recent evidence form single cell RNA seq. Conclusion(s): Here we show that the heterogeneity of dermal fibroblasts observed in scRNAseq is conserved following clonal expansion in vitro. Our novel approach can be exploited to dissect the dynamic changes in fibroblast populations observed within the natural history of SSc skin evolution and ultimately lead to identify appropriate targets to re-establish skin homeostasis or target specific fibroblast populations ultimately limiting toxicity of antifibrotic approaches.