Figure 1.
Synergistic anti-tumor effect of rh-endostatin and adoptive CIK cells therapy on tumor growth.
BALB/C nude mice were injected s.c. with A549 lung cancer cells and when tumor volumes reached 100 mm3, mice were divided into four groups randomly and received respective protocols according to the treatment schema. SPC-A1 and Lewis lung carcinoma xenografts were established and given similar treatments. A, synergistic anti-tumor effect was shown when rh-endostatin was combined with CIK adoptive therapy in suppressing A549 tumor growth (p<0.05). Rh-endostatin or CIK therapy alone did not significantly suppress tumor growth. B, rh-endostatin exhibited a substantial antitumor effect when given in combination with CIK cells in inhibiting SPC-A1 lung carcinoma (p<0.05). C, synergistic anti-tumor effect was shown when rh-endostatin was combined with CIK adoptive therapy in suppressing Lewis lung carcinoma growth (p<0.05). Points, means of tumor volumes of mice per group; Bars, SE. Results shown are representative of three independent experiments.
Figure 2.
Rh-endostatin decreases microvascular density (MVD) and promotes vessel normalization of A549 lung carcinoma.
A549 tumor-bearing mice were treated with rh-endostatin (5 mg/kg, s.c.) for consecutive 7 days with normal saline as control. On days 3, 6 and 9, mice (n = 4, each group and each time point) were sacrificed and tumor samples were stained by anti-CD31 antibody and anti-α-SMA antibody. A, typical fluorescence images of tumors showed CD31-positive endothelial cells (red), α-SMA positive pericytes (green) and merged images (orange) in A549 lung carcinoma in control group and rh-endostatin treated group on days 3, 6, and 9, respectively. B, columns represented microvascular densities at different time points in two groups. C, columns represented pericyte coverages at different time points in the two groups. Representative sections are shown from all groups with a magnification of 400×. Columns, mean; Bars, SE; *p<0.05, indicating significant difference.
Figure 3.
Rh-endostatin treated tumors show increased Ktrans value for Gd-DTPA and delayed Evans blue extravasation.
DCE-MRI and intravital microscopy were performed to test tumor vascular permeability to small molecules and macromolecules, respectively. A, representative images of DCE-MRI with Gd-DTPA well infiltrating in the A549 tumor. B, the columns represent mean ± SE of Ktrans for A549 tumors in control group and rh-endostatin treated group at day3, 6 and 9. C, the columns represent mean ± SE of Evans blue extravasation time in control group and rh-endostatin treated group at day 3, 6 and 9. Columns, mean; Bars, SE; *p<0.05 indicating statistical significance. Results shown are representative of 3 independent experiments.
Figure 4.
Tumor vascular is normalized by rh-endostatin assessed by intravital microscopy.
A549 tumor-bearing mice were treated with rh-endostatin (5 mg/kg, s.c.) for consecutive 7 days with normal saline as control. On days 3, 6 and 9, intravital microscopy were performed to test tumor vascular permeability to macromolecules. A, representative figures showing the exposure of tumor surface and intravenous injection of Evans blue in to BALB/c mice. B, a representative figure of Evans blue infused tumor vessels of control group at 100× magnification. C, a representative figure of Evans blue infused tumor vessels of rh-endostatin treated group at 100× magnification.
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.
Figure 6.
Lymphocytes accumulation in the tumor or spleen of tumor bearing mice.
A, A549 tumor-bearing mice were treated with normal saline or rh-endostatin (5 mg/kg, s.c.) for 7 days. CFSE-labeled 2×107 CIK cells were transfused i.v. into 4 mice from each group on d6 (n = 4). Twenty four hours after CIK cells transfusion, mice were sacrificed and single cell suspensions of spleen and tumor tissue were prepared. CIK cells infiltration was analyzed by flow cytometry. Flow cytometry analysis data showing the percentages of CFSE-labeled CIK cells infiltrating into the tumor and spleen after the administration of normal saline or rh-endostatin. B, the columns represent mean ± SE of CFSE-labeled CIK cells infiltrating percentages into the tumor and spleen of the hosting mice in two groups. C, C57BL/6 mice were injected s.c. with Lewis lung carcinoma cells and the treatment protocols were initiated when tumor volume reached 100 mm3. On day 14, mice were sacrificed and tumor sections were prepared and analyzed by CD3 staining. Ten individual fields surrounding the apoptotic area at 400× magnification were chosen to enumerate the numbers of intratumoral CD3+ T lymphocytes. D, representative sections are shown from all groups at 400× magnification. E, histological H&E staining showing tumor necrosis from each group at 200× magnification. Columns, mean; Bars, SE; *p<0.05 indicating statistical significance.
Figure 7.
Rh-endostatin depletes the accumulation of MDSCs in the tumor.
C57BL/6 mice were injected s.c. with Lewis lung carcinoma cells and when tumor volume reached 100 mm3 treatment were initiated. After administration of rh-endostatin for consecutive 7 days, tumor-bearing mice were sacrificed and single cell suspensions of spleen, lymph node and tumor tissue were prepared to analyze frequency of MDSCs by flow cytometry. A, representative flow cytometry analysis data showing the frequency of CD11b+Gr1+ MDSCs in two groups. B, bar graph depicting the percentages of MDSCs in the spleen, lymph node or the tumor. Columns, mean; Bars, SE; *p<0.05 indicating statistical significance.