Establishment and characterisation of metastatic breast cancer cells expressing fluorescent biomarkers. (360G-Wellcome-202182_Z_16_Z)

Metastasis is the primary cause of cancer deaths. Once cancer cells have disseminated from their original location and formed "secondaries", the only therapeutic option is chemotherapy, which is toxic, ineffective and merely extends patient survival. The prognosis of patients who present with "triple negative breast cancer" (TNBC), which lack the three most common receptors targeted in BC therapy (HER2, progesterone and oestrogen), is extremely poor. TNBC cells disseminate aggressively, typically colonising lymph nodes, lungs, bones, and liver. To better understand this lethal process we are engineering a 3D bioreactor to grow TNBC cells in a relatively "native" state, and will image metastasis live in this system using fluorescence sheet microscopy. To maximise information obtained from our system we need to develop TNBC-sublines expressing fluorescently-labelled marker proteins. Certain structures are indicative of particular behaviours, and can be identified by the presence of specific proteins. Here we will use vinculin to visualise focal adhesions (FA) and assess substrate adhesion; cortactin to identify invadopodia- specialised FAs that invade new tissue; and utrophin as an F-actin surrogate, the principle cytoskeletal component that facilitates cell locomotion. The goal of this project is to generate MDA-MB-23 lines stably expressing mcherry and/or GFP-labelled vinculin, cortactin and utrophin.