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

Figure 3. IRISOE cells entrained by MSCs recruit and activate TAMs.

Figure 3

(A) CCL2 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 level of CCL2 level detected by ELISA in CM of HME, IRIS291, IRIS292, or IRIS293 cells or CM from these cells reconditioned (24h) by MSCs contact. (C) Normalized CCL2 level detected by ELISA in CM of normoxic or hypoxic IRIS291, IRIS292 or IRIS293 cells reconditioned (24h) by MSCs contact. (D) Normalized level detected by ELISA of CCL2 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 CCR2 level on the surface of IRIS291, IRIS292, and IRIS293 cells (upper), or the surface of THP1-macrophages unexposed [-] or exposed to IRIS291, IRIS292, or IRIS293 CM reconditioned by MSCs contact (24h, middle), and same assay performed with normoxic or hypoxic original mammary cells CM (lower). (F-H) Fluorescent IHC staining for CCR2 and the mouse macrophage specific marker F4/80 in 1º IRISOE orthotopic tumor. Scale bars: 200μm in F-H. (I) Recruitment of THP1-macrophages towards CM from IRIS291, IRIS292 or IRIS293 cells reconditioned by MSC contact (24h) in the absence or presence of CCL2 NeuAb detected using Boyden chambers.