Session Item

Fibroblasts undergo premature terminal differentiation after irradiation in vitro associated with increased synthesis and deposition of collagen which is considered important for the development of subcutaneous fibrosis after radiotherapy. In a previous microarray study, we demonstrated down-regulation of cell-cycle-related genes and upregulation of genes involved in extracellular matrix (ECM) deposition and reorganisation, signaling, and inflammation [1]. The purpose of the present study was to validate the expression kinetics over a post-irradiation period of 1-6 days using a different platform.

Early passage skin fibroblast strains (GS3, GS4, GS5) from completely anonymised individual donors were grown in AmnioMax medium as described [1]. RNA was isolated at daily intervals on day 1-6 after irradiation with a single dose of 4 Gy (6 MV X-rays) and analysed on Breakthrough 20k human expression microarrays with ‘dye-swap’ replicates. SAM analysis was done with ‘R’ and JMP Genomics software, and pathway enrichment analysis with the ReactomePA library and ‘Reactome’ pathways. Validation of selected genes and time points in independent experiments was performed with quantitative real-time PCR (qPCR) and Western blotting.

The majority of significant genes showed steady up- or downregulation with increasing time after irradiation. Cell-cycle related genes showed marked downregulation on day 1 and further declined more slowly at later time points. Very few genes showed early upregulation on day 1 with little further increases whereas several genes showed a strong late upregulation with steeper increases from day 2 to 4. Pathway analysis confirmed downregulation of pathways related to cell cycle progression and mitosis as well as upregulation of pathways related to extracellular matrix deposition and remodeling. Signalling pathways (including Interleukin-4 and Interleukin-13 signallng) were overrepresented on day 2 but not on day 5 supporting the notion of a pro-fibrotic response leading to increased ECM deposition. Although most genes showed similar changes in the three individual fibroblast strains, COL11A1 showed larger differences between the three fibroblast strains consistent with the previous study [1], indicating that variations in response of individual donors’ fibroblasts can be detected in vitro.

The results broadly validated and extended the findings from the previous study [1] and supports the use of early-passage fibroblasts as a model for studying fibrogenesis in vitro. Ref. [1]: Herskind et al., Front Cell Dev Biol 9:539893 (2021).