Nitroaspirin corrects immune dysfunction in tumor-bearing hosts and promotes tumor eradication by cancer vaccination

Abstract

Active suppression of tumor-specific T lymphocytes can limit the immune-mediated destruction of cancer cells. Of the various strategies used by tumors to counteract immune attacks, myeloid suppressors recruited by growing cancers are particularly efficient, often resulting in the induction of systemic T lymphocyte dysfunction. We have previously shown that the mechanism by which myeloid cells from tumor-bearing hosts block immune defense strategies involves two enzymes that metabolize L-arginine: arginase and nitric oxide (NO) synthase. NO-releasing aspirin is a classic aspirin molecule covalently linked to a NO donor group. NO aspirin does not possess direct antitumor activity. However, by interfering with the inhibitory enzymatic activities of myeloid cells, orally administered NO aspirin normalized the immune status of tumor-bearing hosts, increased the number and function of tumor-antigen-specific T lymphocytes, and enhanced the preventive and therapeutic effectiveness of the antitumor immunity elicited by cancer vaccination. Because cancer vaccines and NO aspirin are currently being investigated in independent phase I/II clinical trials, these findings offer a rationale to combine these treatments in subjects with advanced neoplastic diseases.

Fig. 1.

NO aspirin restores T lymphocyte function inhibited by myeloid suppressors. (A) Normal splenocytes stimulated in MLR by alloreactive cells are inhibited by MSCs. NCX 4016 restores the proliferative response. Data are expressed as the percentage of proliferation detected in control MLR without MSCs and drugs. The lowest drug concentration that did not negatively affect the proliferation of T lymphocytes in control cultures is reported. (B) Pooled splenocytes from BALB/c tumor-bearing mice were stimulated with alloreactive cells. Results are shown as the fraction of lytic units₃₀ measured in the MLR from tumor-free mice in the presence of the various inhibitors. (C) Splenocyte proliferation induced by anti-CD3 and anti-CD28 is inhibited by MSCs and restored by different NO aspirin isomers. Data are expressed as the cpm means (± SE) of triplicates. SNAP, S-nitroso-N-acetylpenicillamine; GED, guanidinoethyldisulfide bicarbonate; MnTBAP, manganese-(III)-tetrakis-(4-benzoic-acid)-porphyrin. (D) Alloreactivity is restored in tumor-bearing mice by oral or i.p. administration of NCX 4016 or NCX 4060, respectively. Cytotoxicity by MLR-derived lymphocytes from tumor-free mice (no tumor) or control mice treated with NCX 4017 by gavage is shown for comparison.

Fig. 2.

NO aspirin treatment inhibits NOS and ARG activity in vivo.(A) Effect of oral administration of NCX 4016 and NCX 4017 on ARG and NOS activity in CD11b⁺ splenocytes isolated from tumor-bearing mice. Data are from three separate experiments and given as mean ± SE. (B) The immunohistochemical findings on C26-GM tumors taken from NCX-4016-treated and untreated (no NCX) mice were in line with the loss of NOS2 and ARG activity shown in A. NOS2 and nitrotyrosine (in red), largely present in untreated mice tumors, were almost absent in treated mice.

Fig. 3.

Oral administration of NO aspirin recovers the number and function of tumor-specific CD8⁺ T cells induced by cancer vaccine. (A) The absolute number of AH1-TET-positive cells was calculated among gated CD3/CD8-positive cells after subtraction of the background staining obtained by negative control β-Gal_876–884-L^d/TET. (B) Function of peptide-stimulated T lymphocytes was evaluated by IFN-γ release. The values of IFN-γ release are reported as the difference between the values obtained in the presence of peptide-pulsed and unpulsed 293-L^d cells used as stimulators in a 24-h assay. Data are represented as mean ± SE (n = 5).

Fig. 4.

Adjuvant effect of oral NO aspirin on recombinant cancer vaccines. (A) Oral NO aspirin enhances the DNA vaccine coding for an endogenous retrovirus (pcDNA3-env) to prevent challenge with a colon carcinoma. Only the survival rates of mice vaccinated with pcDNA3-env and treated with NCX 4016 differed significantly (Mantel–Haenszel test, P < 0.01) from untreated controls. (B) Oral NO aspirin enhances the therapeutic effectiveness of DNA vaccination on established carcinomas. Only mice treated with the combination NCX 4016 and p185-encoding DNA vaccine showed a significant prolongation of survival (Mantel–Haenszel test, P < 0.01). Control age-matched mice and mice that had rejected the tumor were challenged in the contralateral flank and omolateral superior limb with N2C or CT26 cells, respectively, 70 days after the DNA vaccination (arrow).