Abstract A68: Tumor-associated neutrophils (TAN) develop protumorigenic properties during tumor progression.
Authors: Mishalian Inbal , Bayuh Rachel , Zolotarov Lida , Levy Liran , Singhal Sunil , Albelda Steven M. , Fridlender Zvi Gregorio 
Published: August 2014

Abstract
Introduction: It is becoming increasingly well recognized that tumor develops a complex immunosuppressive network that can paralyze the effector arm of the immune system. We and others have shown that key players in this immunosuppressive network are the tumor associated neutrophils (TAN). TAN can have antitumorigenic (‘N1’) or protumorigenic (‘N2’) function. However, there is limited work on the roles and characteristic of TAN during tumor progression.
Aims: We hypothesized that the phenotype of TAN is dependent on the stage of tumor development, with neutral or anti-tumorigenic, N1-like phenotype in early tumor development, and tumor-promoting, N2-like phenotype in established tumors. We aimed to further characterize TAN's phenotype at different time points during tumor development.
Material and methods: Neutrophil's phenotype was evaluated at early (7 day) and late (14 day) stages after tumor injection, by comparing their properties to each other and to naïve bone marrow (BM) neutrophils. We used neutrophils depletion, Tumor cytotoxicity assays, H202/NO production assays, FACS analysis, immunostaining and RT-PCR to revel the complexities and characteristic of TAN during tumor development.
Results: We found that TAN were more capable of killing tumor cells ex-vivo at early stages of tumor development than TAN from late stages. Our data suggests some possible mechanisms for this ability - production of H2O2, NO and possibly expression of TNF-alpha by TAN from early stages. Gene expression analysis revealed that TAN possesses a “mixed” phenotype, expressing both N2-associated genes (CCl17, ARG, IL-10) and N1-associated genes (CXCL9, ICAM-1, iNOS). However it seems that TAN from established tumor express high levels of N2-associated genes which support tumorigenesis. We found, using Ly6G immunostaining, that at late stages more neutrophils are localized in the central of the tumors, intercalated between tumor cells, whereas at early stages TAN are mainly localized in the periphery of the tumor. Systemic neutrophil depletion after tumor establishment resulted in significantly reduced tumor growth, whereas depletion at early stage of tumor development did not show such an effect.
Conclusions: Our results show that TAN at early stages of tumor growth have more of an N1 phenotype. They are more cytotoxic to tumor cells both directly and indirectly. Later in tumor-growth they are nested in the tumor, acquiring a more “N2-like” phenotype, hence supporting tumor growth. Understanding the effect of tumor on neutrophils, as well as the way these cells support or fight cancer will help to develop strategies to direct the immune system against the tumor.
Citation Format: Inbal Mishalian, Rachel Bayuh, Lida Zolotarov, Liran Levy, Sunil Singhal, Steven M. Albelda, Zvi Gregorio Fridlender. Tumor-associated neutrophils (TAN) develop protumorigenic properties during tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A68.