Combining Antiangiogenic Therapy with Adoptive Cell Immunotherapy Exerts Better Antitumor Effects in Non-Small Cell Lung Cancer Models
Figure 5
Rh-endostatin decreases tumor hypoxic area and hypoxia inhibits the activity of CIK cells in vitro.
A549-bearing mice were divided into two groups and were given rh-endostatin or normal saline respectively for 7 days. On days 3, 6 and 9, mice (n = 4) in the two groups were administrated with pimonidazole. Tumor hypoxic areas were stained by monoclonal antibody (Mab1) against protein adducts of pimonidazole. Hypoxic areas were stained dark yellow. In vitro experiments were conducted by culturing CIK cells under normoxia or hypoxia for 48 h. Proliferation of CIK cells were measured by counting cells with a hemocytometer. A549 lung cancer cells were cocultured with the indicated number of CIK cells under normoxia or hypoxia for 48 h in 96-well plates. The killing rate of CIK cells against the A549 lung cancer cell was measured by LDH release assay. Transmigration assay was performed with Transwell™ inserts containing HUVECs culture. CIK cells were added upon the HUVECs layer and were incubated under normoxic or hypoxic conditions for 48 h. CIK cells that migrated into the lower chamber were harvested and counted by hemocytometry. A, Individual fields at 40×magnification were chosen to represent hypoxic areas in tumor samples on days 3, 6 and 9 in two groups. B, representative pictures of CIK cells cultured under normoxia or hypoxia at 100×magnification. C, bar graphs depicting the density of CIK cells cultured under normoxia or hypoxia. D, bar graphs depicting hypoxic fraction in rh-endostatin treated group or control group. E, bar graphs depicting the cytotoxicity of CIK cells towards A549 cells at different E-to-T ratio. F, bar graph depicting the number of CIK cells migrating across the HUVECs layer. Similar results were observed in three independent experiments. Columns, mean; Bars, SE; *p<0.05 indicating statistical significance.