Abstract 4986: Myeloid-derived suppressor cells as a biomarker of tumor growth and radiosensitivity: Role of hypoxia-inducible arginase-1.

Abstract

Abstract Introduction: Recent evidence suggests that tumor growth and metastases are sustained by myeloid-derived suppressor cells (MDSC), which over-express arginase-1 (ARG) and cause T-cell suppression. We hypothesize that MDSC impair tumor cell radiosensitivity through ARG-mediated depletion of L-arginine, an essential substrate for the biosynthesis of the endogenous radiosensitizer nitric oxide (NO). Aim: We explored whether MDSC and ARG can be used as biomarkers for tumor growth in experimental mouse models and in the clinical setting of colorectal cancer (CRC). We further examined whether low L-arginine levels are associated with an impaired generation of NO by inducible NO synthase (iNOS) and a reduced radiosensitivity of hypoxic tumor cells. Materials & methods: MDSC (CD11b+Gr-1+) and ARG levels in mouse colon CT26 and mammary 4T1 models were analyzed by flow cytometry (FCM) and RT-PCR. Mice were inoculated I.M. with either CT26 or 4T1 carcinoma cells. After 17 days, MDSC were quantified in the spleen by FCM. In addition, ARG levels were analyzed by RT-PCR and FCM. Similar analyses were performed on human blood MDSC (CD14+HLA-DRlow) derived from CRC patients. To model the hypoxic tumor microenvironment, mouse splenocytes were cultured in 1% oxygen. Activation of iNOS was induced by LPS/IFN-γ and analysed by RT-PCR, Western blot and Griess assay. Radiosensitivity was analysed in a model of metabolic hypoxia by colony formation assay Results: The growth of both CT26 and 4T1 tumors in BALB/c mice was associated with an accumulation of MDSC in the spleen from 2-4% (in naive mice) to 8 and 35% respectively. Freshly isolated splenocytes and MDSC showed low ARG levels. Contrasting, hypoxia-conditioned splenocytes from tumor-bearing mice revealed a drastic transcriptional activation of ARG (up to 525.000-fold), and ARG+ MDSC. On the other hand splenocytes from tumor-free mice did not display ARG activation in hypoxic conditions. The iNOS/NO-mediated radiosensitization of mammary EMT6 tumor cells was strictly dependent on L-arginine levels, with a marginal effect of 1.2-fold at 10-30 μM while approaching a 2.4-fold effect at a physiological concentration of 125 μM. MDSC levels in CRC patients were increased, as compared with healthy donors (8-12% versus 4-5%). Patients with locally advanced CRC had 16-fold higher ARG expression in blood monocytes, as compared to healthy donors. 4 weeks after pre-operative radiotherapy the ARG levels decreased by 2-fold. Conclusions: Although tumor progression is clearly associated with the expansion of MDSC, their hypoxic conditioning appears to be crucial to uncover ARG as a tumor biomarker in vivo. iNOS/NO mediated radiosensitisation of hypoxic tumor cells was drastically impaired at low L-arginine levels which could be depleted by ARG+ MDSC. In a clinical setting, we consider ARG a potential biomarker in locally advanced rectal cancer. Citation Format: Wim Leonard, Inès Dufait, Heng Jiang, Femke Steenbeke, Marieke Vermeersch, Kalun Law, Guy Storme, Joeri Aerts, Valeri Verovski, Mark De Ridder. Myeloid-derived suppressor cells as a biomarker of tumor growth and radiosensitivity: Role of hypoxia-inducible arginase-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4986. doi:10.1158/1538-7445.AM2013-4986