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. 2013 May 3;110(22):E1974–E1983. doi: 10.1073/pnas.1216989110

Fig. 4.

Fig. 4.

Biological characterization of 3D nodules cultured under flow vs. nonflow. (A) Compared with nonflow, 3D ovarian cancer nodules grown under flow exhibited morphological features indicative of increased EMT and a more motile phenotype, as observed by DIC and two-photon autofluorescence microscopy and on-chip immunofluorescence staining of a clinically relevant biomarker, EGFR. (B) Fractal dimension (a ratio describing how an object fills space out to its maximum radius with a value of 1 describing a line and a value of 2 a circle) was used to quantify the morphological features of 3D tumors grown under flow versus nonflow conditions. Fractal dimension was significantly lower under flow (1.78 ± 0.01, n = 8 fields) than in nonflow (1.87 ± 0.01, n = 6 fields) conditions (P < 0.05), supporting the observation that flow induces morphological changes consistent with increased EMT in 3D tumors. *** P < 0.001. (C) A significant down-regulation of E-cadherin and CDC2 gene expression was induced by flow (1.0 ± 0.10 and 1.0 ± 0.14, respectively) compared with nonflow cultures (1.5 ± 0.16 and 2.0 ± 0.25, respectively) (n = 6) (P < 0.05). EGFR gene expression levels were not significantly different between nonflow (1.1 ± 0.17) and flow (1.2 ± 0.12) conditions (n = 6), whereas a significant increase in p27Kip1 gene levels was observed under flow (5.3 ± 0.71) relative to nonflow cultures (1.0 ± 0.17) (n = 6) (P < 0.05). *** P < 0.001. (D) In the presence of flow, a significant increase in EGFR expression and phosphorylation was observed (0.70 ± 0.09 and 0.86 ± 0.10, n = 6 and 4, respectively) as compared with nonflow cultures (0.28 ± 0.06, and 0.51 ± 0.09, n = 6 and 4, respectively) (P < 0.05). E-cadherin protein expression decreased significantly in flow (0.19 ± 0.04) as compared with nonflow 3D cultures (0.45 ± 0.10) (n = 9) (P < 0.05), and vimentin expression increased significantly under flow (1.14 ± 0.19, n = 9) compared with nonflow (0.11 ± 0.04, n = 8) conditions (P < 0.05). A flow-induced down-regulation of β-catenin expression was observed (0.67 ± 0.10 under flow compared with 1.3 ± 0.10 in nonflow cultures) (n = 10) (P < 0.05). There was a trend toward a reversal of the protein expression of p27Kip1 and CDC2 (and activation for CDC2), relative to the gene expression for these markers, as a consequence of flow. ***P < 0.001, **P = 0.001 to <0.01, *P = 0.01 to <0.05.