The 3D spheroid model histologically, structurally and transcriptionally resembles fibroblastic foci found within human lung fibrogenesis. It can produce highly crosslinked, collagen similar to that seen in fibrotic lung tissue; so can be used to investigate mechanisms leading to the production of mature, bone-type (pyridinoline) cross-links and the resulting tissue stiffness, both disease hallmarks. This can be used to investigate therapeutics which seek to target collagen crosslinking and tissue stiffness, allowing direct measurement of total collagen, total crosslinks and associated tissue stiffness. We have used this to demonstrate the key role lysyl oxidase-like 2 (LOXL2) has in producing highly crosslinked, stiff extracellular matrix, as well as demonstrating the inability of AB0023, a putative antifbrotic targeting LOXL2, to affect this collagen crosslinking and associated tissue stiffness.

A long-term 3D spheroid model for integrated multi-parameter in vitro studies of fibrogenesis
A. Total collagen normalised to total protein
B. Immature and C. mature collagen cross-links at 2, 4 and 6 weeks culture time
D. Lysyl oxidase family gene expression in spheroid model
E and F. Massan’s Trichrome staining of 6-week E. 3D spheroid model and F. IPF tissue

G. TEM Imaging of collagen fibrils produced by the 3D spheroid model in the presence or absence of the broad-spectrum LOX/LOXL inbitor
H. Images of 3D spheroid models stained with picrosirius red and imaged under plane-polarised light, showing collagen fibrils fluorescence

Tissue stiffness
I. Microsquishing of 3D spheroid model, allows measurement of tissue stiffness
J. LOXL2 inhibition using PXS-S2A and LOX/LOXL inhibition using BAPN strongly reduce spheroid stiffness

Dr Joseph Bell is a research fellow at the University of Southampton.