Figure 4

Cytokine microarray and zymography analysis of CM from 2D and 3D Matrigel model. (A) Comparison of cytokine microarray membranes from different growth conditions of HMF3s and MCF7S1 cells. The cytokines altered in 3D culturing conditions in HMF3s or MCF7S1 cells are numbered and correspond to: 1, GCP-2; 2, IL-6; 3, MCP-1; 4, MCP-3; 5, Amphiregulin; 6, Fas/TNFRSF6; 7, FGF-4; 8, GCSF; 9, GRO; 10, GRO-α; 11, IL-8; 12, MSP-α; 13: TIMP-1, 14, TIMP-2; 15, uPAR (urokinase plasminogen activator receptor); 16, VEGF (vascular-endothelial growth factor); 17, SDF-1; 18, ENA-78; 19, IL-6R; 20, sTNF-RI. (B) Significant increase in cytokine levels in CM from 3D Matrigel co-culture of HMF3s and MCF7S1 cancer cells compared with 3D mono-cultures (upregulated cytokines are marked on the membranes from co-culture). (C) Gelatine zymography showing upregulation of MMP-2 activity. The lower panel shows quantification of band intensities. (D) Western blot analysis of CM from 3D Matrigel co-culture of HMF3s and MCF7S1 cell aggregates showing marked upregulation of MMP-2. The lower panel shows quantification of band intensities of both pro- and active-MMP2. (E) 3D invasion assay stimulated with IL-6 in MCF7S1 monocultures and inhibited with IL-6-specific inhibitor in MCF7S1 + HMF3s co-cultures. A co-culture without the inhibitor is shown for reference.