Fig. 5

Podocalyxin stimulates collective breast tumor cell invasion in 3-D spheroid culture. MCF-7 cells were forced to form aggregates in 3-D culture by first plating them overnight on Matrigel. The aggregates were then overlaid with type I collagen in the presence of EGF for the times indicated. a The MCF-7-control cell aggregates grew considerably in size between day 1 and day 4 and remained relatively spherical. The MCF-7-podo cell aggregates also grew in size but, in addition, they became elongated as they protruded and collectively pushed into the matrix. Live phase microscopy; scale bar = 100 μm. b The collective invasion of the aggregates was quantified after 4 days in 3-D culture by determining their “elongation index”, which consisted of the longest length to width ratio for each aggregate. ***p <0.001, unpaired Student’s t test. c Cell aggregates maintained for 4 days in 3-D culture were fixed and coimmunostained for podocalyxin (red) and E-cadherin (green). Single x/y-axis confocal images (top panels; scale = 40 μm) and 3-D reconstructed images (bottom panels) are shown. The reconstructions show that podocalyxin promotes the cohesive elongation of the aggregates without disrupting E-cadherin localization at cell–cell adhesions. Note also that podocalyxin primarily localized at multiple microlumina within cohesive tumor cell bud-like structures that bulged out from the elongated cell aggregates (arrows). In the lower panels the white outlined box indicates the rotation of the reconstructed confocal stack; the long axis of each box is 211 μm in length