CA 19-9 and β-catenin expression in HT29 spheroids. (A) HT29 spheroids were immunostained against CA 19-9 (red, upper panel) or β-catenin (red, lower panel) and nuclei were counterstained with TOPRO-3 (blue). Acquisitions were taken at 70 μm depth. HT29 spheroids displayed a heterogeneous CA 19-9 expression with a subpopulation of 28% of CA 19-9 negative cells while cytoplasmic, nuclear and membrane associated β-catenin was found in virtually all cells. White arrowheads show nuclear β-catenin immunostain. Scale bar = 50 μm (whole spheroid) and 25 μm (zooms). (B) Permeabilized cells from dissociated HT29 spheroids were labelled against CA 19-9 (upper histogram) or β-catenin (lower histogram) and analysed by flow cytometry. All HT29 spheroid cells were positive for β-catenin whereas 26% of cells were negative for CA 19-9. (C) HT29 spheroids were embedded using the Cytoblock method and stained with the anti-CA 19-9 antibody (upper image) or the anti-β-catenin antibody (lower image) using conventional immunohistochemistry and counterstained with hematoxilin. A subset of cells did not express CA 19-9 (49%) while β-catenin staining was observed in nearly all cells, mainly associated with plasma membrane but also within cytoplasm and nuclei.