Fig. 5.

Interplay between NSCLC cells and HUVECs promotes chemoresistance in MCTSs. a and b NCI-H460 (a) and A549 (b) spheroids were co-cultured with or without stromal cells (WI38 or HUVECs) for 2 days, and then treated with 10 or 20 μM Gefitinib for 2 days. Chemoresistance was measured by staining with EthD-1, a cell death marker. The intensity of EthD-1 was analyzed and values were normalized to control (0.01% DMSO). c and d NCI-H460 (c) and A549 (d) spheroids were co-cultured with or without WI38 cells and HUVECs, and stained with EthD-1 to detect cell death, 2 days after treatment with 10 or 20 μM Cisplatin. Data are shown as mean ± SD from two independent experiments in triplicate. *p < 0.05 versus Control (NCI-H460 or A549). e and f NCI-H460 cells were co-cultured with HUVECs in a 3D culture system, and then treated with 1 μM CHIR-99021, 10 μM or 20 μM Gefitinib (e) or Cisplatin (f) alone, or a combination of both, for 24 h. Cells were harvested and immunoblotted with anti-cleaved PARP, anti-CD31, anti-VE-cadherin, anti-α-SMA, anti-vimentin, and anti-β-actin antibodies. (g) H1975 cells were co-cultured with HUVECs under 3D conditions, and then treated with 1 μM CHIR-99021, 10 μM or 20 μM Gefitinib alone, or a combination of both, for 24 h