Supplementary MaterialsSupplementary File. 0.05, test). ( 0.05, test). ( 0.05, test). ( 0.05, test). Error bars represent SD from at least three independent experiments. Stem-like characteristics were assessed in CD24+CD133+, CD24+CD133?, and CD24?CD133? cells isolated by flow cytometry of human HCC specimens. To determine whether CD24+CD133+ HCC cells were more tumorigenic than CD24?CD133? cells, purified cells were inoculated s.c. into nonobese diabetic (NOD)/SCID mice. A significantly higher tumor incidence was observed at 3 Tipifarnib inhibitor wk after CD24+CD133+ injection compared with injection with CD24?CD133? cells (Fig. 1and and = 12) cells compared with CD24?CD133? (= 40) cells derived from human tumors (Fig. 2 0.05, test). Open in a separate window Fig. 2. iNOS promoted CD133+CD24+ HCC cell tumor initiation and self-renewal capacity in vitro and in vivo. (= 40 and = 12, respectively; * 0.05, test). (= 3; * 0.05, test). (= 6)]. (= 12; * 0.05, test). The higher expression of iNOS in CD24+CD133+ cells leads us to hypothesize that iNOS contributed to the stemness properties in CD24+CD133+ LCSCs. Lentiviral (Lv)-based Tipifarnib inhibitor shRNA-mediated knockdown of iNOS (and shows that as few as 10 CD24+CD133+ LCSCs transfected with scrambled shRNA could form tumors, whereas tumor formation required 1 103 CD24+CD133+ LCSCs transfected with iNOS shRNA. Moreover, the volume of tumors derived from iNOS shRNA cells was lower than that derived from scrambled shRNA cells. IHC and Western blots showed that tumors derived from iNOS shRNA cells exhibited less TACE and NICD than tumors derived from cells that received scrambled shRNA cells (Fig. 2= 24 per group) via the portal vein, and then monitored weekly for bioluminescent signals. To eliminate iNOS activity in the cancer microenvironment, half of iNOS shRNA and control group recipient mice (= 12) received the iNOS inhibitor BYK191023 (31) (60 mg/kg) twice daily starting 1 wk after engraftment. Bioluminescent signals in livers from the BYK191023 group, iNOS shRNA group, or iNOS shRNA with BYK191023 group were weaker throughout the observation period than those from Tipifarnib inhibitor the control group (Fig. 2= 12). The incidence of tumor formation was 100% in the untreated control CD24+CD133+ group, but it was reduced to 66.7%, 50%, and 25% in the BYK191023, iNOS shRNA, and iNOS shRNA with BYK191023 groups, respectively (= 12). Collectively, these data suggest that iNOS in both the LCSCs and the tumor microenvironment promote CD24+CD133+ HCC cell tumor initiation and raise the possibility that iNOS-directed therapeutics may represent an effective LCSC-targeted strategy for inhibiting tumor Tipifarnib inhibitor growth. iNOS Promotes the CSC Phenotype and Tumorigenicity via Activating Notch1. A recent study demonstrated that NO enhances glioma stem cell self-renewal capacity (17). Therefore, we investigated whether NO could drive the self-renewal and tumorigenicity of CD24+CD133+ LCSCs by overexpressing iNOS/NO with an adenovirus vector (Ad)-iNOS or LV-iNOS vector (and 0.05, test). ( 0.05, test). Our previous study demonstrated that the Notch pathway is activated DKFZp564D0372 in LCSCs and inhibition of the Notch pathway in CSCs suppresses tumorigenicity, cell invasion, and migration (33, 34). We next analyzed the link between iNOS and Notch pathway activation in Ad-iNOSCtransduced CD24+CD133+ LCSCs. CSL-luciferase reporter/promoter constructs, which can be activated by Notch signaling, were transiently transfected into CD24+CD133+ MHCC-97H cells. Overexpression of iNOS significantly increased luciferase activity in the CSL-luciferaseCexpressing cells relative to untreated control LCSCs derived from MHCC-97H and PLC/PRF/5 HCC cells (Fig. 3 0.01). Transduction with Ad-iNOS also led to an increase in mRNA levels for the Notch1 receptor and the Notch target gene Hes1 relative to controls (Fig. 3 0.05, test). RLU, relative luciferase activity. (and = 0.001 and 0.001, respectively). Cox regression analysis showed that CD24 [hazard ratio (HR) = 2.355, = 0.009], iNOS (HR = 2.028, = 0.0013), active TACE (HR = 2.482, 0.0001), and NICD (HR = 2.487, = 0.0076) are independent predictors of 7-y survival after adjustment for age at diagnosis, sex, TNM stage, and neoadjuvant therapy (= 0.001 and ** 0.001, respectively). (and = 0.478, ** 0.001) and activated TACE (Pearson = 0.4604, ** 0.001). (= 0.025] and advanced TNM stage (OR = 3.72, = 0.034), high NICD expression correlated significantly with the presence of microsatellites (OR = 4.66, = 0.05), venous infiltration (OR = 14.24, = 0.0006), and advanced TNM stage (OR = 3.37, = 0.048), after adjustment for age at diagnosis, sex, and neoadjuvant therapy. Table 1. Association of high NOS2 expression with tumor characteristics = 90)NICD (= 90)OR.