Abstract 5600: Macrophage activation and differentiation augment immunotherapy of established mouse lung tumors
Authors: Fridlender Zvi G. , Jassar Arminder , Kapoor Veena , Cheng Guanjun , Sun Jing , Albelda Steven M. 
Published: February 2011

Abstract
Background: An immunosuppressive environment exists within tumors, induced by both cancer and immune cells, that inhibits the effect of cytotoxic T-lymphocytes. 5,6-Dimethylxanthenone-4 acetic acid (DMXAA) is a small flavanoid-like compound, previously shown to have a strong anti-tumor activity in murine models of cancer, and currently being tested in phase 3 trials in humans. We have shown that part of the effect induced by DMXAA as monotherapy is by activating tumor-associated macrophages. Given the ability of DMXAA alone to potentially induce a tumor microenvironment conducive to anti-tumor immune responses, we hypothesized that it would be a useful adjunct to immuno-gene therapy (immunoRx) in lung cancer.
Methods: We injected 18 mg/kg i.p. of DMXAA (Sigma) to tumor-bearing animals and evaluated its impact in three immunoRx models of non-small cell lung cancer (NSCLC): two aimed at an expressed HPV-E7 antigen, using an adenovirus (Ad. E7) or a Listeria (Listeria. E7) vector, and one using an adenovirus expressing Interferon-α. We compared the effect of immunoRx alone to combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, changes in macrophage phenotype, and tumor microenvironment. This was done by real time RT-PCR, flow cytometry, and staining (IHC).
Results: DMXAA markedly augmented the effect of immuno-gene therapy on large (&gt;200 mm3) established NSCLC flank tumors in the three models and two cell lines of NSCLC tested, resulting in cures in 30-40% of mice. We evaluated mechanisms in the Ad. E7 model. There was no significant difference in the subtypes of systemic (Splenic) immunocytes between the two groups. In contrast, DMXAA increased the intra-tumoral influx of neutrophils, macrophages, and CD8+ T-cells compared to the immunoRx group. Furthermore, adding DMXAA to immunoRx decreased the percentage of intra-tumoral M2 (CD206+) macrophages (out of all tumor cells) by 2-fold, while increasing the percentage of M1 macrophages by 2-fold. Although the percentage of CD8+ T-cells was increased in tumors with the combined treatment compared to immunoRx alone, as seen both by flow cytometry and IHC, there was no difference in their activation or E7 antigen-specificity. They were no changes in the influx of T-regulatory cells. Adding DMXAA to immunoRx up-regulated mRNA levels for a host of pro-inflammatory and chemo-attractant cytokines in tumors including TNF-α, IFN-γ, IL-10, CCL-5 and CCL2.
Conclusion: Macrophage activation and differentiation using DMXAA significantly augments the efficacy of immunoRx in established murine NSCLC tumor models by increasing the traffic of immunocytes into the tumors, activating and differentiating macrophages to a more anti-tumor phenotype and creating an immunostimulatory microenvironment. Our data suggests that combining agents like DMXAA with vaccines should be considered in future immunoRx trials.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5600.