12th Annual Symposium
Physics of Cancer
Aug 30 - Sept 1, 2021
|PoC - Physics of Cancer - Annual Symposium|
All bark and no bite?: Studying the role of YAP in a novel, stiffness-dependent multicellular tumour spheroid invasion model with high- and super-resolution imaging
Randall Centre for Cell and Molecular Biophysics, King's College London, Newcomen Street, London, UK
Multicellular tumour spheroids embedded within collagen matrices are a powerful approach for studying invasion and cell-ECM interactions in a versatile manner, with flexibility found in collagen concentration and labelling as well as in differing spheroid sizes or compositions as well as standard biochemical approaches also being utilised such as drug treatments and siRNA. However, given the difficulty in achieving consistent sample preparation, imaging methods are often limited by sample depth and density. To overcome these limitations, we have developed a versatile, micro-fabricated PDMS stamping-based method for consistently embedding spheroids in 3D collagen matrices at a specific position in 3D space to allow for high- and super-resolution imaging in an in vivo-like setting. We have used our method to test the effect of the mechanosensory protein YAP on collective cell invasion from MCF7 and HCC1954 breast cancer spheroids in collagen matrices in a stiffness-dependent manner. Upon YAP inhibition or knockdown, with verteporfin and siRNA respectively, we see a decrease in cell invasion over a 5 day time period compared to controls. Formalin fixation and IF of embedded spheroids shows increased YAP activity in the outer spheroid cells. Collagen staining allows us to image cell-ECM interactions, with significant collagen alignment between HCC1954 spheroids, while rapid high-/super-resolution imaging of multicellular protrusions with the SoRa spinning disk system enables quantification of actin-based protrusions such as filopodia. Super-resolution imaging of filopodia reveals a stiffness-dependent relationship affecting filopodia density and length, while knockdown of YAP with siRNA indicates a role for YAP in filopodia formation. Our method also permits a variety of other applications, such as ECM ablation, spheroid FLIM and study of spheroid-spheroid interactions. In conclusion, this novel method enables reliable imaging of cell-ECM, cell-cell, whole spheroid and inter-spheroid dynamics at a range of magnifications over long periods of time with great flexibility provided by a medley of staining methods, plating in various sample holders and more.