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Mooney et al. J Cancer Metastasis Treat 2019;5:19 I http://dx.doi.org/10.20517/2394-4722.2018.93 Page 11 of 17

A B

C D

E F

Figure 6. Western blot analysis of long-term protein expression of key canonical Wnt/β-catenin signaling pathway molecules. Metastatic
MDA-MB-231 breast cancer cells (BCCs) were co-cultured with embryonic stem cell (ESC)-microstrands for 48 h, followed by removing
ESC-microstrands, replacing BCC media, and analyzing protein expression at 72 h and 120 h. Western blots for protein expression in
metastatic MDA-MB-231 BCCs (CC: co-cultured with ESC-microstrands; E: co-cultured with empty microstrands; C: non-co-cultured
control) (A); densitometry of expression of β-catenin (B), NKD2 (C), precursor E-cadherin (D), mature E-cadherin (E), and vimentin (F)
(***P < 0.001; **P < 0.01; *P < 0.05)

E-cadherin present in the BCCs co-cultured with ESC-microstrands, and it was extremely elevated [Figure
6D]. Mature E-cadherin was expressed in the BCCs co-cultured with ESC-microstrands, but not in the non-
co-cultured controls at 72 h. and 120 h [Figure 6E]. There was some expression in the empty microstrand
control at 72 h, however, it was much less. Our previous work demonstrated that co-culture with ESC-
[10]
microstrands decreases vimentin protein expression after 48 h . At 72 h and 120 h, vimentin protein
expression was diminished in MDA-MB-231 BCCs co-cultured with ESC-microstrands [Figure 6F].

DISCUSSION
This work has demonstrated the utility of a bioengineered 3D ESC microenvironment (so-called ESC-
microstrands) for the study of cross-talk of signaling pathways in cancer cells. It reveals that inhibitory
effects of the ESC microenvironment on triple negative, metastatic MDA-MB-231 BCCs is attributable to
restoration of EGFR and canonical Wnt/β-catenin signaling pathway regulation. Simultaneous treatment of