Fig. 7

Stable knockdown of podocalyxin in highly invasive 4T1 murine breast tumor cells reduces collective invasion. a Whole cell lysates from syngeneic murine breast tumor cell lines with different capacities to metastasize were probed for endogenous podocalyxin expression. Endogenous podocalyxin was most highly expressed in 4T1 cells, which are highly metastatic compared with the nonmetastatic 67NR and weakly metastatic 66 cl4 cell lines [42]. b 4T1 cells stably transfected with either control vector (4T1-control) or vectors containing an shRNA sequence targeting the 3′ untranslated region of the murine podocalyxin transcript were analyzed for endogenous podocalyxin expression by western blotting. Stable 4T1 cell populations, each expressing a different short hairpin sequence, show a moderate (podo KD-3293) and near complete knockdown of endogenous podocalyxin (podo KD-3603), respectively. c 4T1-control or 4T1-podo KD 3603 tumor cells were aggregated on Matrigel overnight, overlaid with collagen type I, and maintained in 3-D culture for 4 days. They were then imaged, live, by phase microscopy (upper panels) or fixed and immunostained for endogenous podocalyxin (red) and the tight junction protein ZO-1 (green) followed by confocal imaging (lower panels). The 4T1-control cells invaded as cohesive multicellular strands with scattered small pockets of poorly polarized podocalyxin throughout. In contrast, aggregates from podocalyxin knockdown cells did not effectively invade as cohesive multicellular strands; they did, however, occasionally release single cells into the matrix (arrowheads). Scale = 50 μm