RESUMO
Background: Cancer cells remodel their local physical environment through processes of matrix reorganisation, deposition, stiffening and degradation. Urokinase-type plasminogen activator (uPA), which is encoded by the PLAU gene, is an extracellular proteolytic enzyme known to be involved in cancer progression and tumour microenvironment (TME) remodelling. Perturbing uPA therefore has a strong potential as a mechano-based cancer therapy. This work is a bioengineering investigation to validate whether 1) uPA is involved in matrix degradation and 2) preventing matrix degradation by targeting uPA can reduce cancer cell invasion and metastasis. Methods: To this aim, we used an engineered 3D in vitro model, termed the tumouroid, that appropriately mimics the tumour's native biophysical environment (3 kPa). A CRISPR-Cas9 mediated uPA knockout was performed to introduce a loss of function mutation in the gene coding sequence. Subsequently, to validate the translational potential of blocking uPA action, we tested a pharmacological inhibitor, UK-371,801. The changes in matrix stiffness were measured by atomic force microscopy (AFM). Invasion was quantified using images of the tumouroid, obtained after 21 days of culture. Results: We showed that uPA is highly expressed in invasive breast and colorectal cancers, and these invasive cancer cells locally degrade their TME. PLAU (uPA) gene knock-out (KO) completely stopped matrix remodelling and significantly reduced cancer invasion. Many invasive cancer gene markers were also downregulated in the PLAU KO tumouroids. Pharmacological inhibition of uPA showed similarly promising results, where matrix degradation was reduced and so was the cancer invasion. Conclusion: This work supports the role of uPA in matrix degradation. It demonstrates that the invasion of cancer cells was significantly reduced when enzymatic breakdown of the TME matrix was prevented. Collectively, this provides strong evidence of the effectiveness of targeting uPA as a mechano-based cancer therapy.
RESUMO
Naturally derived materials are often preferred over synthetic materials for biomedical applications due to their innate biological characteristics, relative availability, sustainability, and agreement with conscientious end-users. The chicken eggshell membrane (ESM) is an abundant resource with a defined structural profile, chemical composition, and validated morphological and mechanical characteristics. These unique properties have not only allowed the ESM to be exploited within the food industry but has also led to it be considered for other novel translational applications such as tissue regeneration and replacement, wound healing and drug delivery. However, challenges still exist in order to enhance the native ESM (nESM): the need to improve its mechanical properties, the ability to combine/join fragments of ESM together, and the addition or incorporation of drugs/growth factors to advance its therapeutic capacity. This review article provides a succinct background to the nESM, its extraction, isolation, and consequent physical, mechanical and biological characterisation including possible approaches to enhancement. Moreover, it also highlights current applications of the ESM in regenerative medicine and hints at future novel applications in which this novel biomaterial could be exploited to beneficial use.
