NO, Immunosuppression and Tumor Immmunotherapy

This study aims at clarifying the role of NO in the immunosuppression induced by in vivo tumor growth and by tumor immunotherapy, and determining whether the inhibition of NO production can be used as an adjuvant in tumor immunotherapy. We have shown previously that tumor cells, glioma (N32) and colon carcinoma (H1D2), when genetically engineered to express such immune stimulatory cytokines as IFN-g and IL-18, induce strong anti-tumor immune response, in immunized tumor-free rats, whereas only a limited therapeutic effect is achieved in rats in which a tumor has already become established. This led us to our studying whether immmunosuppression induced in rats by a subcutaneously growing malignant glioma and in rats with an intrahepatic growing colon carcinaoma would attenuate anti-tumor immune responses. Anti-tumor cytolytic responses, proliferative responses and cytokine production were found to all be strongly suppressed in the spleen cells of these tumor-bearing rats. The suppression was also shown to be partially dependent on the production of nitric oxide (NO) by the suppressor cells found in the plastic adherent fraction of the spleen cells. Since during inflammatory responses the major part of NO is derived from the IFN-g induced expression of inducible nitric oxide synthase (iNOS), it was of interest to investigate whether the expression of iNOS is induced after immunization by IFN-g-secreting glioma cells (N32 IFN-g) and whether the inhibition of NO generated by iNOS in immunized rats would enhance anti-tumor immune responses. The expression of iNOS was found to be elevated, both at the immunization site and in the brain tumors. The selective inhibition of iNOS was shown to enhance anti-tumor immune responses. In conclusion, our results demonstrate that in immunized rats both growing tumors and immunotherapeutic intervention by use of IFN-g secreting tumor cells tends to induce NO-dependent suppressor cell activity that inhibits anti-tumor immune responses. It appear then that the selective inhibition of iNOS can be used as an adjuvant for enhancing type 1 anti tumor T-cell responses during anti-tumor immunotherapy

Enhanced expression of iNOS intratumorally and at the immunization site after immunization with IFNgamma-secreting rat glioma cells

Nitric oxide (NO) can modulate both tumor growth and antitumor immune responses. In order to elucidate the mechanism of curative therapeutic immunization with IFNgamma-producing glioma cells, we examined the expression of inducible nitric oxide synthase (iNOS) in tissue sections from immunized animals. There was a significantly enhanced iNOS expression both intratumorally and at the immunization site. Although the mechanisms behind this dual expression of iNOS most probably are different, our results suggest a role for NO in both the induction and execution of the antitumor response

Inhibition of Inducible Nitric Oxide Synthase Enhances Anti-tumour Immune Responses in Rats Immunized with IFN-gamma-Secreting Glioma Cells.

Interferon gamma (IFN-gamma) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN-gamma has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN-gamma (N32-IFN-gamma), could enhance the anti-tumour immune response. Initially, both a selective iNOS, L-N-6-(I-Iminoethyl)-L-lysine (L-NIL), and non-selective, N-nitro-L-arginine methyl ester (L-NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both L-NIL and L-NAME enhanced proliferation and production of IFN-gamma from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, L-NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32-IFN-gamma), and administered NOS inhibitors by intraperitoneal (i.p.) mini-osmotic pumps, only splenocytes of rats treated with L-NIL, but not L-NAME, displayed an enhanced proliferation and production of IFN-gamma when re-stimulated with N32 tumour cells. Based on these findings, L-NIL was administered concurrently with N32-IFN-gamma cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN-gamma-based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects

Numb protein expression correlates with a basal-like phenotype and cancer stem cell markers in primary breast cancer.

Decreased expression of Numb, resulting in activation of the proto-oncogene Notch1 and reduction in the tumor suppressor p53, has been demonstrated in mammary carcinomas. The aim of this study was to investigate the relationship between Numb protein expression and clinicopathological characteristics, tumor biological subtypes and putative cancer stem cell markers in a well-characterized cohort of primary human breast cancers. Immunohistochemistry was performed on tissue microarrays of primary invasive breast tumors using a polyclonal anti-Numb primary antibody. Of the 241 tumors evaluated, 50 (21%) displayed deficient or reduced Numb immunoreactivity. Retained Numb expression was significantly correlated to estrogen (ER) and progesterone receptor (PR) positivity (P < 0.001 and P = 0.004, respectively). Interestingly, we found that a higher percentage of the tumors with deficient or reduced Numb expression belonged to the triple-negative (ER-/PR-/HER2-) subgroup compared to tumors with retained Numb expression (P = 0.004). Transcriptional profiling of a subset of these tumors linked NOTCH1 and BIRC5, both downstream targets of Numb, to the triple-negative subgroup in an inverse manner. Typically, subgroups characterized by the low expression of Numb expressed higher levels of NOTCH1 and BIRC5 (encoding survivin). We also found deficient expression of Numb in a significantly higher proportion of BRCA1 dependent tumors, which are usually triple-negative, compared to sporadic tumors. The expression of Numb in 14 breast cancer cell lines correlated similarly to their respective molecular subtypes. We further established an inverse correlation between the Numb expression levels and the CD44+/CD24- cancer stem cell phenotype (P = 0.05) in primary tumors. Finally, decreased Numb expression was associated with poorer distant disease-free survival (P = 0.01). Taken together, our results indicate that loss of Numb expression is a marker of tumor aggressiveness, potentially linked to BRCA1 status and a cancer stem cell phenotype in primary breast cancer