Activated macrophages as a novel determinant of tumor cell radioresponse: the role of nitric oxide-mediated inhibition of cellular respiration and oxygen sparing.

PURPOSE: Nitric oxide (NO), synthesized by the inducible nitric oxide synthase (iNOS), is known to inhibit metabolic oxygen consumption because of interference with mitochondrial respiratory activity. This study examined whether activation of iNOS (a) directly in tumor cells or (b) in bystander macrophages may improve radioresponse through sparing of oxygen. METHODS AND MATERIALS: EMT-6 tumor cells and RAW 264.7 macrophages were exposed to bacterial lipopolysaccharide plus interferon-gamma, and examined for iNOS expression by reverse transcription polymerase chain reaction, Western blotting and enzymatic activity. Tumor cells alone, or combined with macrophages were subjected to metabolic hypoxia and analyzed for radiosensitivity by clonogenic assay, and for oxygen consumption by electron paramagnetic resonance and a Clark-type electrode. RESULTS: Both tumor cells and macrophages displayed a coherent picture of iNOS induction at transcriptional/translational levels and NO/nitrite production, whereas macrophages showed also co-induction of the inducible heme oxygenase-1, which is associated with carbon monoxide (CO) and bilirubin production. Activation of iNOS in tumor cells resulted in a profound oxygen sparing and a 2.3-fold radiosensitization. Bystander NO-producing, but not CO-producing, macrophages were able to block oxygen consumption by 1.9-fold and to radiosensitize tumor cells by 2.2-fold. Both effects could be neutralized by aminoguanidine, a metabolic iNOS inhibitor. An improved radioresponse was clearly observed at macrophages to tumor cells ratios ranging between 1:16 to 1:1. CONCLUSIONS: Our study is the first, as far as we are aware, to provide evidence that iNOS may induce radiosensitization through oxygen sparing, and illuminates NO-producing macrophages as a novel determinant of tumor cell radioresponse within the hypoxic tumor microenvironment.

IFN-gamma+ CD8+ T lymphocytes: possible link between immune and radiation responses in tumor-relevant hypoxia.

Activated T lymphocytes are known to kill tumor cells by triggering cytolytic mechanisms; however, their ability to enhance radiation responses remains unclear. This study examined the radiosensitizing potential of mouse CD8+ T cells, obtained by T-cell-tailored expansion and immunomagnetic purification. Activated CD8+ T cells displayed an interferon (IFN)-gamma+ phenotype and enhanced by 1.8-fold the radiosensitivity of EMT-6 tumor cells in 1% oxygen, which modeled tumor-relevant hypoxia. Radiosensitization was counteracted by neutralizing IFN-gamma or by blocking the inducible isoform of nitric oxide synthase, thus delineating the immune-tumor cell interaction through the IFN-gamma secretion pathway. Reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorter data in agreement detected downregulation of the IFN-gamma gene by hypoxia, which caused IFN-gamma deficiency next to radioresistance. Therefore, immune and radiation responses are likely to be allied in the hypoxic tumor microenvironment, and CD8+ T cells may bridge immunostimulatory and radiosensitizing strategies.

The radiosensitizing effect of immunoadjuvant OM-174 requires cooperation between immune and tumor cells through interferon-gamma and inducible nitric oxide synthase.

PURPOSE: To explore whether antitumor immunoadjuvant OM-174 can stimulate immune cells to produce interferon-gamma (IFN-gamma) and thereby radiosensitize tumor cells. METHODS AND MATERIALS: Splenocytes from BALB/c mice were stimulated by OM-174 at plasma-achievable concentrations (0.03-3 mug/mL), and afterward analyzed for the expression and secretion of IFN-gamma by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Stimulated splenocytes were used as a source of IFN-gamma to radiosensitize hypoxic EMT-6 tumor cells through the cytokine-inducible isoform of nitric oxide synthase (iNOS). RESULTS: OM-174 activated the production of IFN-gamma at high levels that reached 70 ng/mL in normoxia (21% oxygen) and 27 ng/mL in tumor-relevant hypoxia (1% oxygen). This caused up to 2.1-fold radiosensitization of EMT-6 tumor cells, which was associated with the iNOS-mediated production of the radiosensitizing molecule nitric oxide, as confirmed by accumulation of its oxidative metabolite nitrite, Western blot analysis, and reverse transcriptase-polymerase chain reaction. Both iNOS activation and radiosensitization were counteracted by neutralizing antibodies against IFN-gamma. The same mechanism of radiosensitization through the IFN-gamma secretion pathway was identified for IL-12 + IL-18, which are known to mediate IFN-gamma responses. Hypoxia displayed a dual effect on the immune-tumor cell interaction, by downregulating the expression of the IFN-gamma gene while upregulating iNOS at transcriptional level. CONCLUSION: Immunoadjuvant OM-174 is an efficient radiosensitizer of tumor cells through activation of the IFN-gamma secretion pathway in immune cells. This finding indicates a rationale for combining immunostimulatory and radiosensitizing strategies and extends the potential therapeutic applications of OM-174.

Macrophages enhance the radiosensitizing activity of lipid A: a novel role for immune cells in tumor cell radioresponse.

PURPOSE: This study examines whether activated macrophages may radiosensitize tumor cells through the release of proinflammatory mediators. METHODS AND MATERIALS: RAW 264.7 macrophages were activated by lipid A, and the conditioned medium (CM) was analyzed for the secretion of cytokines and the production of nitric oxide (NO) through inducible nitric oxide synthase (iNOS). EMT-6 tumor cells were exposed to CM and analyzed for hypoxic cell radiosensitivity. The role of nuclear factor (NF)-kappaB in the transcriptional activation of iNOS was examined by luciferase reporter gene assay. RESULTS: Clinical immunomodulator lipid A, at a plasma-relevant concentration of 3 microg/mL, stimulated RAW 264.7 macrophages to release NO, tumor necrosis factor (TNF)-alpha, and other cytokines. This in turn activated iNOS-mediated NO production in EMT-6 tumor cells and drastically enhanced their radiosensitivity. Radiosensitization was abrogated by the iNOS inhibitor aminoguanidine but not by a neutralizing anti-TNF-alpha antibody. The mechanism of iNOS induction was linked to NF-kappaB but not to JAK/STAT signaling. Interferon-gamma further increased the NO production by macrophages to a level that caused radiosensitization of EMT-6 cells through the bystanding effect of diffused NO. CONCLUSIONS: We demonstrate for the first time that activated macrophages may radiosensitize tumor cells through the induction of NO synthesis, which occurs in both tumor and immune cells.

Lipid a radiosensitizes hypoxic EMT-6 tumor cells: role of the NF-kappaB signaling pathway.

PURPOSE: Lipid A has shown promising immunostimulatory effects in both experimental tumor models and advanced stage cancer patients. This study examines whether lipid A may directly modulate the radioresponse of tumor cells by activating inducible nitric oxide synthase (iNOS) or cyclooxygenase-2 (COX-2) through nuclear factor-kappaB (NF-kappaB) signaling. METHODS AND MATERIALS: Hypoxic EMT-6 tumor cells were exposed to lipid A and analyzed for the level of COX-2 and iNOS by Western blotting and enzymatic assays. The hypoxic radioresponse of EMT-6 cells was estimated by clonogenic survival. The activation of NF-kappaB was examined by immunostaining of its p65 subunit and by luciferase reporter gene assay. RESULTS: Lipid A dose-dependently increased the expression and activity of iNOS with a maximal effect at plasma achievable concentrations of 3-30 micro g/mL. The COX-2 mediated production of prostaglandin E2 was constitutively high and further upregulated by lipid A. The radiosensitivity of hypoxic EMT-6 cells was increased up to 2.5 times and counteracted by the iNOS inhibitor aminoguanidine but not by the COX-2 inhibitor NS-398. The mechanism of radiosensitization was linked to NF-kappaB signaling, because its inhibition by phenylarsine oxide impaired both iNOS activation and radioresponse. CONCLUSION: Lipid A is an efficient hypoxic cell radiosensitizer at plasma relevant concentrations, which provides a rationale to combine lipid A with radiotherapy in further studies.