Immunohistochemistry: Bond System(Leica Microsystems, Milton Keynes, UK). Antibodies will have specificity confirmed by Western blotting with positive and negative controls. Antibodies will be optimized with positive control cell blocks and oesophageal tissue before application to primary tumours and lymph nodes. PCR for oncogenic mutations: DNA will be extracted from serial sections from formalin fixed paraffin. Select gene mutations (single nucleotide variants, SNVs) which are frequently occurring and known to be relevant to lymph node metastases from our own ongoing work and from the literature. Bioinformatics support to robustly pick deleterious SNVs and verify these within the same cases as part of the ICGC project. PCR will be performed using standard techniques and high thoroughput systems using fluidigm platforms for analysis of multiple SNVs in parallel. Cell Lines: We have a panel of cell lines derived from oesophageal adenocarcinoma including 2 lines from metastases. These have been verified either in house or as part of an international effort to verify the lines for use in in vitro model systems. We will use standard sRNA methods to knockdown targets of interest or to overexpress them using standard transfection methods. Proliferation (promega Glo assay), apoptosis (FACS analysis, caspase expression) and invasion through matrigel will be determined using standard methods, already established in the laboratory for other targets. Site-directed mutagenesis may also be used to recapitulate the mutation of interest. Lymphangiogenesis: Human microvascular lymphatic endothelial cells will either be co-cultured with tumour cells or tumour conditioned medium for 24 hours. The ability of tumour cells to invade and home specifically to lymphatic vessels and lymph nodes rather than blood vessels will be determined using a combination of modified Boyden chamber and 3D co-culture models. Long-term imaging of cultures will be performed on a Leica SP5 confocal microsc