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. Author manuscript; available in PMC: 2019 May 15.
Published in final edited form as: Clin Cancer Res. 2018 Mar 2;24(10):2370–2382. doi: 10.1158/1078-0432.CCR-17-2545

Figure 3. Chemotherapy induces monocyte expression of MCPs, which promote the stemness-associated properties by inducing Notch.

Figure 3

(A & B) Co-cultures were set up using MDA-MB-231 or BT474 BC cells with monocytes isolated from C57BL/6 mice treated with DOXO, DTX, or PBS, or with DOXO/DTX/PBS-pretreated THP-1 cells, in the presence or absence of the CCR2 inhibitor MK-0812 (A) or CCL2/7/8 NAb (B) as described in Materials and Methods. BC cells and monocytes were seeded at a ratio of 1:1 or 1:3. ALDEFLUOR assays (for MDA-MB-231) and ESA+CD44+CD24−/low flow cytometry (for BT474) were performed using BC cells harvested after 48 h of co-culture. (C) MCP secretion by THP-1 and CAF was determined by ELISA of the conditioned media (CM) of 105 THP-1 cells or cancer-activated CAF (pre-treated with the CM from PDX265922 cancer cells) that had been treated with DOXO (125 nM), DTX (4 nM), or PBS for 48 h. (D) THP-1 cells under DOXO/DTX/PBS treatment and mouse monocytes isolated as in A were cultured in the presence or absence of a JNK inhibitor SP600125 (1 μM) for 48 h and analyzed by quantitative RT-PCR using GAPDH/Gapdh for normalization. (E) MDA-MB-231 cells were treated with CCL2/7/8 at the indicated concentrations for 48 h and analyzed by sphere formation assay or ALDEFLUOR assay. (F) PDX265922 cells derived from a primary TNBC were treated with CCL2/7/8 (1 ng/ml) or PBS for 48 h and analyzed by sphere formation assay or ALDEFLUOR assay. (G) CCL2/7/8-treated BT474 cells were analyzed by sphere formation assay or flow cytometry for the ESA+CD44+CD24−/low population. (H) Indicated BC cells were treated with CCL2/7/8 (1 ng/ml) or PBS for 24 h and analyzed by quantitative RT-PCR for indicated stemness-related genes. Data are normalized to GAPDH and compared to the PBS group. (I) Western blot analysis showing stemness-associated gene expression in BC cells treated with CCL2/7/8 or PBS for 24 h. (J) Western blot analysis of MDA-MB-231 cells treated with CCL2/7/8 or PBS in the presence or absence of a γ-secretase inhibitor (GSI) RO4929097 (10 μM). *P<0.05, **P<0.01, ***P<0.001 (compared to the control group in the first column of each group or as indicated).