Characterization of iNOS+ Neutrophil-like ring cell in tumor-bearing mice

Lauren P Virtuoso,Nejat K Egilmez,Jamie L Harden,Wade J Sigurdson,Fuminobu Yoshimura,Boris Minev,Paula Sotomayor,Mehmet O Kilinc

doi:10.1186/1479-5876-10-152

Abstract

BackgroundMyeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset.MethodsSingle-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS+ ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS+ ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro.ResultsThe results showed that intracellular iNOS staining distinguished a granular iNOS+ SSChi CD11b+ Gr-1dim F4/80+ subset with ring-shaped nuclei (ring cells) among the CD11b+ Gr-1+ cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8+ T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells.ConclusionsIn this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.

Highlights

Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer
Identification of an inducible nitric oxide synthase (iNOS)+ subset among the tumor-resident myeloid cell populations In the tumor microenvironment, Nitric oxide (NO) activity by infiltrating myeloid cells has been suggested to represent a mechanism for their immunosuppressive properties
To determine whether iNOS+ cells constitute a distinct subset among heterogeneous tumor-resident myeloid cell populations, single-cell suspensions from primary tumors were stained for extracellular markers CD11b, Gr-1 and F4/80 followed by intracellular iNOS staining

Summary

Introduction

Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Tumorderived inflammatory factors prevent proper differentiation of IMC resulting in the appearance of a highly heterogeneous myeloid cell population with subsets arrested at different stages of development [2] They are known to be activated by various factors secreted by tumor stroma and produce increased levels of Arginase (ARG) or iNOS, which have been associated with T cell suppression [3]. As evidence by all these recent studies, identification of an individual MDSC subset with a specific function has been difficult because of the lack of unique cell surface markers that can distinguish between different myeloid subtypes Their classification was further complicated by the plasticity of MDSC [reviewed in Refs. An example of the phenotypic switch among the myeloid cell population is that F4/80+ monocytes have been shown to be the precursors of functionally distinct subsets of TAM [9] and DC [13,14]

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