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Page 12 of 17 Mooney et al. J Cancer Metastasis Treat 2019;5:19 I http://dx.doi.org/10.20517/2394-4722.2018.93

Figure 7. Schematic representation of the effects of co-culture with embryonic stem cell (ESC)-microstrands on expression of key
signaling pathway molecules in MDA-MB-231 breast cancer cells (BCCs). Left Panel: BCC alone; Right Panel: co-cultured breast cancer
cells with ESC-microstrands. Changes in protein and mRNA expression after co-culture with ESC-microstrands indicated inhibition of the
canonical Wnt/β-catenin (above the dash line) and EGFR (below the dash line) signaling pathways. Co-culture with ESC-microstrands
causes NKD2 up-regulation at both the mRNA and protein levels, and this coincides with dual signaling pathway inhibition. It suggested
restored ability of NKD2 to inhibit the canonical Wnt/β-catenin signaling pathway and reduced ability to promote the EGFR signaling
pathway following co-culture

metastatic BCCs with EGFR and canonical Wnt/β-catenin signaling inhibitors suppresses growth more than
individual treatment, suggesting that both pathways play a role in the restriction of metastatic phenotype.

The microenvironments of ESCs play a fundamental role in providing cells with appropriate signaling
to induce cell proliferation, differentiation, or death. ESCs secrete soluble factors or exosomes that could
reprogram malignant cancer cells to benign phenotype and suppress tumorigenesis [35-39] . To recapitulate
the native stem cell niche, researchers have exploited several bioengineering methods. Researchers have
demonstrated that human ESC pluripotency is maintained when the ESCs are cultured in a 3D hyaluronic
[43]
[40]
acid , alginate [41,42] , or chitosan scaffold . Specifically, our lab has reported that mouse ESCs encapsulated
inside aqueous alginate hydrogel microsbeads remain pluripotent and restrict rat breast cancer cell
[8]
proliferation and migration . Our previous work has also established that ESCs encapsulated in alginate
microstrands exhibited biological effects on cell behavior of BCCs after co-culture with these ESC-
microstrands, restricting highly aggressive human breast cancer proliferation, survival, invasion, migration,
[10]
EMT, and metabolic activity as summarized in Table 1. This study aims to further probe whether this
restriction is due to restored signaling pathway function, specifically related to the canonical Wnt/β-catenin
and EGFR signaling pathways, which are hyperactivated in human breast cancer.