Khan, E. S. | bioRxiv Science

bioRxiv2021

Collagen density defines 3D migration of CTLs and their consequent cytotoxicity against tumor cells

Solid tumors are often characterized by condensed extracellular matrix (ECM). The impact of dense ECM on cytotoxic T lymphocytes (CTL) function is not fully understood. Here, we report that CTL-mediated cytotoxicity is substantially impaired in dense collagen matrices. Although the intrinsic killing machinery including expression of cytotoxic proteins and degranulation was intact, CTL motility was substantially compromised in dense collagen. We found that for 3D CTL migration, persistence and velocity was regulated by collagen stiffness and the porosity, respectively. Interestingly, 3D CTL velocity is strongly correlated with their nuclear deformability/flexibility during migration, which is regulated by the microtubule network. Moreover, CTL migration was completely abolished by inhibition of actin polymerization and or myosin IIA. Remarkably, disruption of the microtubule-networks significantly improves the impaired migration, search efficiency, and cytotoxicity of CTLs in dense collagen. Our work suggests the microtubule network as a promising target to rescue impaired CTL killing capacity in solid tumor related scenarios.

immunology↗

bioRxiv2019

Exogenous supply of Hsp47 triggers fibrillar collagen deposition in skin cell cultures in vitro

BackgroundCollagen is a structural protein that provides mechanical stability and defined architectures to skin. In collagen-based skin disorders like Epidermolysis bullosa, EDS the ability to offer such stability is lost either due to mutations in collagens or defect in the chaperones involved in collagen assembly, which leads to chronic wounds, skin fragility, and blisters. Existing approaches to study and develop therapy against such conditions are the use of small molecules like 4-phenylbutyrate (4-PBA) or growth factors like TGF-{beta}. However, these approaches are not collagen specific resulting in unsolicited responses. Therefore, a collagen specific booster is required to guide the correct folding and deposition of collagen in a highly regulated manner. Hsp47 is a chaperone with a major role in collagen biosynthesis. Expression levels of Hsp47 correlate with collagen production. This article explores the stimulation of collagen deposition by exogenously supplied Hsp47 (collagen specific chaperone) in skin cells, including specific collagen subtypes quantification.\n\nResultsHere we quantify the collagen deposition level and the type of deposited collagens by different cell types from skin tissue (fibroblasts NHDF, L929 and MEF, keratinocytes HaCat and endothelial cells HDMEC) after Hsp47 stimulation. We find upregulated deposition of fibrillar collagen subtypes I, III and V after Hsp47 delivery. Network collagen IV deposition was enhanced in HaCat and HDMECs and fibril-associated collagen XII were not affected by the increased Hsp47 intracellular levels. The deposition levels of fibrillar collagen were cell-dependent i.e. Hsp47-stimulated fibroblasts deposited significantly higher amount of fibrillar collagen than Hsp47-stimulated HaCat and HDMECs.\n\nConclusionsA 3-fold enhancement of collagen deposition was observed in fibroblasts upon repeated dosage of Hsp47 within the first 6 days of culture. Our results provide fundamental understanding towards the idea of using Hsp47 as therapeutic protein to treat collagen disorders.

cell biology↗