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. 2017 Sep 14;8(61):103182–103206. doi: 10.18632/oncotarget.20892

Figure 5. IRISOE cells entrained by MSCs recruit and activate ECs.

Figure 5

(A) VEGF level in HME cells transfected with doxycycline-inducible IRIS allele in the absence (HME) or presence of 2μg/ml of Dox (72h, HME/IRIS). (B) Normalized VEGF level detected by ELISA in CM from HME, IRIS291, IRIS292 or IRIS293 cells, or CM from these cells reconditioned (24h) by MSCs contact. (C) Normalized VEGF level detected by ELISA in CM from normoxic or hypoxic IRIS291, IRIS292 or IRIS293 cells reconditioned (24h) by MSCs contact. (D) The level of VEGF detected by ELISA secreted from naïve MSCs (red bar), HME, IRIS291 or IRIS293 alone (white bars), or in CM from HME, IRIS291 or IRIS293 reconditioned by MSCs contact in the absence (black bars) or presence of IL-1β NeuAb (yellow bars) added before MSCs contact, or CXCL1 NeuAb (green bars) added after MSCs contact. (E) Western blot analysis of VEGFR2 level on the surface of IRIS291, IRIS292, and IRIS293 cells (upper), or the surface of HUVEC unexposed [-] or exposed to IRIS291, IRIS292 or IRIS293 CM reconditioned by MSCs contact (24h, middle), and the same assay performed with normoxic or hypoxic original mammary cells (lower). (F-H) Fluorescent IHC staining for VEGFR2, and the mouse endothelial cells specific marker CD31 in 1° IRISOE orthotopic tumor. Scale bars: 100μm in F-H. (I) Recruitment of HUVEC towards CM from IRIS291, IRIS292, or IRIS293 cells reconditioned by MSC contact (24h) in the absence or presence of VEGF NeuAb detected using Boyden chambers.