Urokinase-type plasminogen activator (uPA) regulates invasion and matrix remodelling in colorectal cancer

Auxtine Micalet; Luke J Tappouni; Katarzyna Peszko; Despoina Karagianni; Ashley Lam; John R Counsell; Sergio A Quezada; Emad Moeendarbary; Umber Cheema

doi:10.1016/j.mbplus.2023.100137

Urokinase-type plasminogen activator (uPA) regulates invasion and matrix remodelling in colorectal cancer

Matrix Biol Plus. 2023 Nov 15:19-20:100137. doi: 10.1016/j.mbplus.2023.100137. eCollection 2023 Dec.

Authors

Affiliations

¹ UCL Centre for 3D Models of Health and Disease, Department of Targeted Intervention, Division of Surgery and Interventional Science, University College London, Charles Bell House, 43-45 Foley Street, London W1W 7TS, United Kingdom.
² Department of Mechanical Engineering, University College London, Gower Street, London WC1E 6BT, United Kingdom.
³ UCL Centre for Targeted Cancer Therapies, Department of Targeted Intervention, Division of Surgery and Interventional Science, University College London, Charles Bell House, 43-45 Foley Street, London W1W 7TS, United Kingdom.
⁴ Immune Regulation and Tumour Immunotherapy Group, UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, United Kingdom.
⁵ 199 Biotechnologies Ltd., Gloucester Road, London W2 6LD, United Kingdom.

Abstract

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.

Keywords: 3D models; CRISPR-Cas9; Cancer invasion; Mechano-based cancer therapy; Stiffness; Tumour microenvironment; Urokinase-type plasminogen activator; uPA.