LGR5 immunoreactivity was marginally elevated in tumours derived from LIM1899 cells transfected with pTune LGR5, but not in all areas of the tumours. LGR5 staining was decreased, but not totally absent, in tumours derived from LIM1899 cells transfected with siLGR5. In these samples, LGR5 expression appeared restricted to the glandular structures. The opposite phenotypes observed after knockdown or overexpression of LGR5, and their consistency in transient and LY2157299 stable expression systems, indicate that this phenomenon is directly correlated to LGR5 levels. Thus LGR5 may modulate the balance between cell-cell and cellsubstrate adhesion. To characterize cell-matrix and cell-cell interactions we cultured cells as spheroids in Temozolomide Hanging drops and performed both wound assays and motility assays to measure the migration potential of the cells. Hanging drops assays measure the proliferative potential of the cells in the absence of cell-matrix interactions; wound assays assess the rate of movement of a cell monolayer, and the motility assay measures the rate of migration of the cells through the ECM in filter pores. Parental LIM1899 cells, or LIM1899 cells transfected with empty vectors, grow in hanging drops as aggregates with dense centres. In parallel cultures, spheroids of cells with reduced levels of LGR5 are surrounded by a halo of loosely-associated cells and are easily disrupted, while cells expressing high levels of LGR5, either transiently or stably, pack into compact spheroids resistant to mechanical disruption. The difference in cell density is reflected in the volume of the spheroids relative to their cellularity: while spheroids from different cell lines contain similar number of cells, the difference in volume between siLGR5 spheroids and spheroids overexpressing LGR5 is significant reflecting a tighter packing of the cells in M2LGR5. In wound repair assays LGR5 overexpressing cells have a reduced ability to repopulate the scratch wound compared to the parental cells, and accumulate at the edge of the wound forming a densely packed ridge of viable cells. Both assays confirm the original observation that LGR5 overexpression favours cell-to-cell adhesion. Transwell assays were used to monitor the migration ability of cells with different levels of LGR5. Cells were seeded in Transwell filters and incubated for three days, before switching the upper filter contents to serum-free medium to encourage migration. Under these conditions, cells with reduced levels of LGR5 migrated to the underside of the filters to a much greater extent than untransfected cells, while cells overexpressing LGR5 had significantly reduced migration. These differences in migrating cell numbers were highly significant: p = 0.002 for siLGR5, p = 0.03 and p = 0.001 for transient and stable overexpressors, respectively. Consistently, the reduction in motility displayed by LGR5 overexpressing cells was proportional to the level of LGR5 expression.