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Tracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo -activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression. Ex vivo -expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg- Prkdcscid IL2rgtm1Wjl/ SzJ (NSG) mice. The authors administered 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry. The fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45+CD56+CD3− NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D. Administered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45+CD56+CD3− NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression. [ABSTRACT FROM AUTHOR]

Background aims Irradiation enhances the adhesion between natural killer (NK) cells and target cells by up-regulating intercellular adhesion molecule-1 (ICAM-1) on target cells. Therefore, we investigated the effect of irradiation-induced ICAM-1 expression on human cancer cells on NK cell–mediated cytotoxicity. Methods Expression levels of ICAM-1 on the target cell surface before and after irradiation of six human cancer cell lines (HL60, SKBR-3, T47D, HCT-116, U937 and U251) were analyzed by flow cytometry. Ex vivo expansion of NK cells from human peripheral blood mononuclear cells was performed by co-culture with irradiated K562 cells. The related adhesion molecule lymphocyte function–associated antigen 1 (LFA-1) on NK cells was analyzed by flow cytometry. An enzyme-linked immunosorbent assay was used to detect interferon-γ (IFN-γ), and WST-8 assays were performed to check NK cell cytotoxicity. Finally, blocking assays were performed using monoclonal antibodies against ICAM-1 or LFA-1. Results LFA-1 expression increased on NK cells after expansion ( P  <   0.001). The expression of ICAM-1 was significantly upregulated by irradiation after 24 h in various cell lines, including HL60 ( P  <   0.001), SKBR-3 ( P  <   0.001), T47D ( P  <   0.001) and U937 ( P  <   0.001), although the level of expression depended on the cell line. ICAM-1 expression was extremely low before and after irradiation in U251 cells. NK cell–mediated cytotoxicity increased after irradiation of HL60 ( P  <   0.001), SKBR-3 ( P  <   0.001), T47D ( P  = 0.003), and U937 ( P  = 0.004) cells, in which ICAM-1 expression was significantly increased after irradiation. IFN-γ production by NK cells in response to HL60 ( P  <   0.001) and T47D ( P  = 0.011) cells significantly increased after irradiation. NK cell–mediated cytotoxicity against irradiated SKBR-3 ( P  <   0.001) and irradiated T47D cells ( P  = 0.035) significantly decreased after blocking of ICAM-1. Blocking of LFA-1 on NK cells resulted in reduced cytotoxicity against irradiated HL60 ( P  <   0.001) and irradiated SKBR-3 ( P  <   0.001). Conclusions Irradiation upregulates ICAM-1 expression on the surface of human cancer cells and enhances activated NK cell–mediated cytotoxicity. Therefore, irradiation combined with NK cell therapy may improve the antitumor effects of NK cells. [ABSTRACT FROM AUTHOR]