In Vitro Generation of TGFb- and IL1a-Induced Fibroblast Subtypes for Mechanistic Studies of Extracellular Matrix Composition and Architecture

Purpose In solid cancers, inflammation and viral infections, two main fibroblastic functions have been identified: myofibro-blast-like fibroblasts and inflammatory fibroblasts. In the tumour microenvironment (TME), these cancer-associated fibro-blast (CAFs) subtypes are known as myCAFs, which generate a stiffened fibrotic extracellular matrix (ECM), and iCAFs,which secrete inflammatory cytokines to locally modulate the immune response. Yet, whether iCAFs contribute to shapingthe ECM biochemical and biophysical properties remains understudied, mainly because robust in vitro models to generatefibroblast subtypes are lacking.Methods Here, we established an in vitro cell culture system based on murine NIH3T3 fibroblasts and stimulation by TGFβ1and IL1α, alone or in combination, to induce fibroblast subtypes. Gene expression analysis of well-documented myCAF(Acta2/Tagln) and iCAF (Ccl2/Il6/Lif) markers was performed. We compared these in vitro subtypes to myCAFs and iCAFsfrom publicly available scRNAseq data of tumour tissues from cancer patients. ECM deposition and remodelling wereassessed by collagen contraction, fluorescence microscopy and atomic force microscopy.Results TGFβ1 induced a myCAF-like phenotype, while a combination of TGFβ and IL1α induced an iCAF-like phenotype.We found that, similar to myCAFs, both tumour-associated and in vitro iCAFs express Acta2/Tagln as well as genes encodingtypical ECM proteins, which correlated in vitro with the ability to contract collagen. Finally, fluorescence microscopy andatomic force microscopy revealed that in vitro both subtypes generate thick, layered and stiff matrices with highly alignedECM.Conclusions These findings demonstrate that, besides myCAFs, iCAFs may also contribute to a pathological ECM. Ourstudy provides new insights into the contribution of IL1α- and TGFβ1-induced fibroblast subtypes to ECM deposition andremodelling and puts forward a well-defined in vitro model to activate different fibroblast functions for future in-depthmechanistic studies of fibroblast subtype-specific roles in cancer and other pathologies.