Study of fibroblasts activation kinetics and identification of fibroblast subpopulations in physiological and pathological situations

Abstract

Fibroblasts undergo significant morphological and functional changes in response to specific environmental cues and functional changes. In wound healing processes and cancerous environments, fibroblasts undergo transformative activation adopting novel phenotype. The aim of this project was to understand the changes in cell morphology and cytoskeletal reorganization that occur for both normal-associated fibroblasts (NAFs) and cancer-associated fibroblasts (CAFs) activation. To achieve this, we used an innovative approach using biophysical biomarkers derived from epifluorescence imaging of the cell's cytoskeleton, employing CSKmorphometrics. Clustering algorithms have been applied to do a preliminarily identification of CAFs subpopulations. Our findings confirm that cytoskeletal reorganization occurs during both physiological and pathological activation. Non-tumoral fibroblasts experience larger morphological changes characterized by an increase in area and cell convexity, as well as changes in the total fluorescence of F-actin fibers during the 24 hours posterior to the administration of TGF-β. On the contrary CAFs exhibited sustained larger areas throughout the process regardless of TGF-β administration. They underwent most drastic changes in fiber length and showed a significant increase in nuclear volume. The application of logistic regression algorithms has allowed for a classification with 81% accuracy to differentiate between CAFs and NAFs, highlighting the differences in the cytoskeleton of these cell types in both study contexts. On the other hand, the intragroup analysis provided by clustering has enabled the identification of 5 clusters for non-activated CAFs, which converge at 72 hours into two larger clusters with significant differences. This study enhances understanding of the changes occurring in CAFs and NAFs during activation from a cytoskeletal point of view and remarks the need for a study of fibroblasts subpopulations as well as the need for novel biomarkers.