The radiosensitizing effect of immunoadjuvant OM-174 requires cooperation between immune and tumor cells through interferon-gamma and inducible nitric oxide synthase.
OBJECTIVE
To explore whether antitumor immunoadjuvant OM-174 can stimulate immune cells to produce interferon-gamma (IFN-gamma) and thereby radiosensitize tumor cells.


METHODS
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.


CONCLUSIONS
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.
De Ridder
Mark
M
Academic Hospital Free University Brussels, Oncology Center, Cancer Research Unit, Brussels, Belgium. Mark.De.Ridder@vub.ac.be
Verovski
Valeri N
VN
Chiavaroli
Carlo
C
Van den Berge
Dirk L
DL
Monsaert
Christinne
C
Law
Kalun
K
Storme
Guy A
GA
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D015536:Down-Regulation; D004789:Enzyme Activation; D007111:Immunity, Cellular; D007371:Interferon-gamma / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018664:Interleukin-12 / Q000378:metabolism; D020382:Interleukin-18 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011836:Radiation Tolerance / Q000187:drug effects / Q000276:immunology; D013154:Spleen / Q000166:cytology; D015854:Up-Regulation
D000276:Adjuvants, Immunologic; D020382:Interleukin-18; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D018664:Interleukin-12; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C400105:defoslimod