pNK and NK-92 cells sensitized tumor cells.

<p>1×10⁵ of various tumor cells were seeded in 96-well tissue-culture plates, co-cultured with 2.5×10⁵ pNK cells for 4 h, washed and then exposed to 800 cGy of irradiation and evaluated 48 h late for cell proliferation by the MTS (A). C, cancer cell alone; C/NK, cancer cell and NK coculture; C+RT, cancer cell treated with 800 cGy radiation; C/NK+RT, cancer cell and pNK coculture followed by radiation. The apoptosis of CNE-1 cells after irradiation 48 h under various co-culture conditions that described previously was analyzed by (B) Annexin-V assay and (C) cell cycle analysis. (D) CNE-1 cells were incubated with NK-92 cells in 1∶2.5 ratios for 2, 4, and 8 h and irradiated at indicated doses. (E) 1×10⁵ CNE-1 cells were cultured in the lower chambers of transwells, and 2.5×10⁵ NK-92 cells were cultured in the upper chambers for 4 and 8 h. Both of (D) and (E) were assayed using Annexin-V to detect apoptotic cells (AnnexinV⁺). (*, <i>p</i><0.05).</p

The caspase signaling pathway was induced after co-culture.

<p>CNE-1 cells were co-cultured with 2.5 fold of NK-92 cells for 4 h, then NK-92 cells were washed away, and CNE-1 cells were exposed to 800 cGy of radiation. Control cells of CNE-1 alone or CNE-1 cells that had been co-cultured were not irradiated. After 24 h of incubation, cells were harvested for western blot analysis of procaspase/caspase-3, procaspase-8, and procaspase-9 protein in lysates of CNE-1 alone (lane C), CNE-1 cells cultures with NK-92 cells (lane C/N). The arrows indicate cleaved (activated) caspase 3 at about 17 kDa and its precursor, pro-caspase 3, at about 43 kDa; precaspase 8, at about 55kDa; procaspase 9, at about 45kDa. β-actin was used as the internal control.</p

Caspase-3 was inhibited by XIAP and XIAP was downregulated by binding of Smac after radiation.

<p>CNE-1 cells (lane C) were treated with NK-92 cells for 4 h (lane C/N) before combined treatment with 800 cGy of radiation. (A) Cell lysates were immunoprecipitated with anti-XIAP antibody and immunoblotted with anti-Smac, anti-caspase-3 or anti-XIAP antibody. (B) CNE-1 cells were transfected with 80 nM of XIAP siRNA for 16 h and co-cultured with NK-92 cells for 4 h before NK-92 cells were washed away. The cells were assayed using Annexin-V to determine the percentage of apoptotic cells (AnnexinV⁺). (C) Cell lysates were immunoprecipitated by anti-Smac antibody and detected with anti-XIAP antibody by Western blot. (D) CNE-1 cells were treated with NK-92 cells for 4 h (C/N) before combined treatment with 800 cGy of radiation (C/N+RT) or CNE-1 treated with 800 cGy of radiation alone (C+RT). After treatment, cells were further incubated for 0 min, 15 min, 2 h, or 24 h, then harvested and fractionated into cytosolic (Cyto) and mitochondrial (Mito) fractions for assay by western blot. β-actin was used as the loading control for each fraction. Density of the XIAP normalized with β-actin was assayed by Image J. The bar chart was average of three independent experiments.</p

Primary NK cells sensitized tumor cells with same pathway.

<p>CNE-1 cells were transfected with 80 nM of XIAP siRNA for 16 h and co-cultured with pNK cells for 4 h before pNK cells were washed away. The cells were assayed using Annexin-V to determine the percentage of apoptotic cells.</p

Effect of NK-92-treated CNE-1 cells on Fas blockage.

<p>CNE-1 cells were co-cultured with 2.5 fold NK-92 cells for 4 h in presence of anti-FasL blocking antibody (10 µg/ml). The percentage of apoptotic cells after irradiation 48 h under various co-culture conditions was analyzed by Annexin-V assay (AnnexinV⁺, D). Results from 3 independent experiments are shown; bars indicate mean ± SD.</p

Mechanism of reciprocal interaction between NK cells and radiation in target cells.

<p>NK cells damage target cell through perforin/granzyme B and death receptor/caspase mediated pathway. The radiosensitisation effect through NK cell depends more on the perforin/granzyme B pathway. Without radiation, the suboptimal activation of NK cells cause up-regulation of XIAP. With radiation, the mitochondria releases Smac to neutralize XIAP and enhances NK cell-mediated cytotoxicity.</p

Additional file 1: Figure A. of Improving immunological tumor microenvironment using electro-hyperthermia followed by dendritic cell immunotherapy

(a) Representative flow cytometric analysis on immature and mature DCs. The purple histograms represent the isotype-matched control, and the red line histograms represent staining with specific antibodies. *MFI represents the mean fluorescence intensity, which are expressed on the right upper corner of each histogram. Error bars represent standard errors. (b) Histogram plot of MFI. (*) P < 0.05, (**) P < 0.01 (t-test) compared with the immature DC. Figure B. Endocytotic activity was measured by flow cytometry in DCs generated from bone marrow cultured for 9 d with 20 ng/ml of GM-CSF; mDCs obtained from iDCs were incubated with 10 g/mL of AH1 and 50 g/mL of Hsp70 for 24 h and reacted with 100 mg/mL of FITC-Dextran at 4°C (purple) or 37 °C (green line) for 2 h before analysis. This result was represented from one of three independent experiments. (DOCX 608 kb