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. 2018 Nov 27;7(12):234. doi: 10.3390/cells7120234

Figure 4.

Figure 4

Identification of the mesenchymal-like “migrating and metastasis initiating” breast cancer stem cell phenotype clone. (A) NRF1+E2 CD24−CD44+ BCSCs were further flow sorted for the isolation of the CD24−CD44+CD49f+ALDH+CXCR4+CD133+. (B) Graph showing NRF1+E2-transformed MCF10A cells displaying CD44+CD49f+ALDH+CXCR4+NRF1+ BCSCs subtype and the most highly aggressive breast cancer cell line—MDA-MB231 (Figure 4A,B). (C–J) Phenotypic characteristics of CD44+CD49f+ALDH+CXCR4+NRF1+ BCSCs subtype assessed by live imaging of tumorosphere formation, the migration and proliferation potential, and xenograft tumor growth assays; large tumor spheroids were observed by HoloMonitoring (C) and confocal microscopy (D), as live images of NRF1 and NRF1+E2 BTIC clones compared to the vector control did not form tumor spheroids at 5 and 15 days. (E,F) This BCSC clone demonstrated a significantly higher potential to close the wound 6 h after initiation of the wound healing assay compared to the vector clone (** p < 0.01 and * p < 0.05) and higher migration capacity compared to vector clone with and without E2 treatment (E–H). (I,J) Effects of anti-estrogens—fulvestrant and tamoxifen; a chemotherapeutic agent—paclitaxel on tumorosphere formation ability of NRF1 overexpressing BTICs or mesenchymal MDA-MB231 enriched with CD44+ +CD133+CXCR4+ clones. (K) Xenograft tumor growth NRF1 overexpressing CD44+CD49f+CD133+CXCR4+ BTIC clone after 42 days in (5 out of 5) mice (p < 0.001). The upper panel shows macroscopic images of resected tumors, the middle panel shows weight with image of tumors, and the lower panel tumor growth curve of the NRF1 BCSC-like subset and inhibition of tumor growth by silencing CXCR4 expression.