Abstract
Breast cancer development is profoundly impacted by physical changes that occur in the tumour microenvironment throughout growth and metastasis of tumours. Extracellular matrix stiffness increases as tumours develop and spread, with stiffer environments thought to increase cancer cell invasion. Changes in extracellular stiffness and other physical characteristics are sensed by integrins which integrate these extracellular cues to intracellular signalling through proteins such as YAP and TAZ. YAP and TAZ are master regulators of mechanotransduction that respond to changes in extracellular stiffness and are also oncogenes involved throughout the metastatic cascade, although the complexities of how YAP and TAZ impact tumour development in variable stiffness environments are currently unclear.Multicellular tumour spheroids can be generated by methods such as hanging drop and embedded in collagen gels to permit study of the processes affecting cancer cell invasion in an in vivo-like setting. However, standard spheroid invasion assay preparation methods often result in inconsistent sample preparation and limited application of advanced imaging techniques. We developed a novel method for optimised high- and super-resolution imaging to examine the role of YAP during MCF7 and HCC1954 breast cancer spheroid invasion by microfabricating custom PDMS stamps to stamp well structures into collagen gels of varying stiffness. Our PDMS stamping method was shown to provide superior image quality and experimental consistency compared to standard spheroid invasion assay preparations.
MCF7 GFP spheroids did not invade into 3D collagen matrices, while HCC1954 lifeact- GFP spheroids and MCF7 spheroids overexpressing GFP-YAP invaded collectively in low stiffness collagen matrices but not in high stiffness matrices. High stiffness matrices reduced cell proliferation and collagen alignment, but increased YAP nuclear localisation. This increased YAP nuclear localisation was shown via multi-photon ablation assays to be largely due to ECM network tension. We verified the role of YAP during spheroid invasion with verteporfin, a small molecule YAP inhibitor, and siRNA-mediated knockdown of YAP, while YAP inhibition reduced cell proliferation. At later invasive timepoints, collectively invading broad multicellular protrusions displayed an increased nuclear YAP localisation. The density and length of filopodia were reduced by YAP knockdown and increased matrix stiffness. YAP knockdown reduced radial collagen alignment and ECM remodelling was shown to be profoundly impacted by MMPs and actomyosin contractility. ML141 treatment, MMPs and actomyosin contractility significantly reduced spheroid invasion, with Cdc42 and MMPs acting synergistically in this context. In conclusion, overexpression and high endogenous expression of YAP have been shown to induce an invasive phenotype in breast cancer spheroids which involves ECM remodelling, MMPs, actomyosin contractility and enhanced protrusiveness. Future work should seek to further clarify the role of these components and others that are generally implicated with YAP and which may underlie observed phenotypes such as Myc, Src and actin remodellers. This experimental system is amenable to an expanded suite of potential approaches such as alternative imaging modalities, use of patient-derived organoids/ECM and modified PDMS stamping techniques which could all be used in future to broaden the scope and in vivo relevance of findings.
| Date of Award | 1 Aug 2022 |
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| Original language | English |
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| Supervisor | Susan Cox (Supervisor) & Madeline Parsons (Supervisor) |