Super resolution microscopy to unravel complex medicine-induced changes in the protein nano environment


Super resolution microscopy to unravel complex medicine-induced changes in the protein nano environment


L Toms1; D Yilmaz1; H Barjat1; R Kelly1; I R Holden2; N R Thomas2; I Peset-Martin1; M Burnham1
1 Medicines Discovery Catapult, UK;  2 University of Nottingham, UK


Super resolution microscopy (STORM), with resolution <30nm, enables visualisation of the cellular nano environment. We have used this technique at the forefront of drug discovery to investigate the mechanism of action of a novel complex medicine nanoparticle. This nanoparticle, a HER2-specific Affibody fused to Apoferritin, aims to improve treatment of HER2+ breast cancers, where current treatment, Trastuzumab, is limited due to resistance. Here, we compared the effects of the Affibody Apoferritin and Trastuzumab in breast cancer cell lines where overexpressed HER2 is resistant to internalisation and drives formation of abnormal membrane protrusions.

HER2+ Breast cancer cell lines were treated with Alexa647 labelled Affibody Apoferritin or Trastuzumab for 24 hours and the number of internalised particles determined using high-content imaging. STORM was used to assess HER2 cluster spatial properties. HER2+ cells were treated for 2 or 24 hours with Affibody Apoferritin or Trastuzumab and labelled for HER2. HER2 clusters were analysed using a density-based clustering algorithm in cell edge (membrane protrusions) and cell body (non-protruding membrane) regions. Dual-colour STORM was used to visualise both treatment and HER2 densities within filopodia.

Affibody Apoferritin was internalised into breast cancer cell lines. After 24 hours, Affibody Apoferritin and Trastuzumab reduced HER2 cluster area at the cell edge. Whereas, in the cell body, Affibody Apoferritin increased cluster area and number of clusters. Strikingly, Affibody Apoferritin had a greatly reduced density ratio towards filopodia compared to Trastuzumab.

This work shows complex medicines acting in the cellular nano environment, identifying potential differences in mechanism at cellular structures of only 100-200nm which are thought to be important drivers of oncogenic transformation. Additionally, this work highlights the capability of new STORM techniques in drug discovery.

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