Evaluation of Myeloid-derived Suppressor Cells and Components of Renin Angiotensin System in Urethaneinduced Lung Cancer

Special Article-Cancer Immunology

J Immun Res. 2015;2(2): 1018.

Evaluation of Myeloid-derived Suppressor Cells and Components of Renin Angiotensin System in Urethaneinduced Lung Cancer

Jair Ribeiro Chagas1,2, Bruna Visniauskas1, Guiomar Nascimento Gomes3 and Valquiria Bueno4*

1Department of Psychobiology, UNIFESP Federal University of So Paulo, Brazil

2Department of Biosciences, UNIFESP Federal University of So Paulo, Brazil

3Department of Physiology, UNIFESP Federal University of So Paulo, Brazil

4Department of Microbiology, Immunology and Parasitology, UNIFESP Federal University of So Paulo, Brazil

*Corresponding author: Valquiria Bueno, Department of Microbiology Immunology and Parasitology, UNIFESP Federal University of So Paulo, Brazil

Received: February 19, 2015; Accepted: March 06, 2015; Published: March 09, 2015

Abstract

Urethane-induced lung cancer has been associated with an imbalance in the effector/suppression immune response since interferon-gamma production is increased along with elevated percentages of myeloid-derived suppressor cells. The goal in cancer therapy is to prevent the immunosuppressive effects caused by tumor and thus lead to the development of effective immune response. The depletion of myeloid-derived suppressor cells in experimental models causes less tumor burden and improves the immune response but doesn´t provide cure. As cancer is a complex and multifactorial disease the treatment also should be based on multiple targets.

A recently described target is the renin angiotensin system and its components (i.e. bradikynin receptors, angiotensin-converting enzyme) as they may play a role in cancer development. Therefore, our aim was to investigate the percentage of myeloid-derived suppressor cells at the tumor site and the possible correlation with bradikynin receptors and angiotensin-converting enzyme expressions in mice submitted to Urethane-induced lung cancer.

BALB/c mice were either injected with myeloid-derived suppressor cells, Urethane or only followed for 120 days (control). Lung, spleen, and blood were phenotyped for myeloid-derived suppressor cells and lung tissue was evaluated for the expression of renin angiotensin system components.

In tumor-bearing mice it was observed a significant increase at the percentage of myeloid-derived suppressor cells in the spleen along with angiotensinconverting enzyme increased gene and decreased protein expressions in lung.

Urethane administration was crucial to lung tumor development, to the increase of peripheral myeloid-derived suppressor cells and to the changes observed in angiotensin-converting enzyme (ACE).

In conclusion, tumor microenvironment seems to modulate myeloid-derived suppressor cells expansion and ACE gene and protein expressions. Myeloidderived suppressor cells and renin angiotensin system components could be potential targets for cancer diagnostics and therapy.

Keywords: Lung cancer; Urethane; Myeloid-derived suppressor cells; Angiotensin-converting enzyme; Bradykinin

Introduction

Our group has shown previously that Urethane-induced lung cancer leads to an increase in the percentage of myeloid-derived suppressor cells (MDSC) in spleen and lung along with transforming growth factor-beta (TGF-β) expression by tumor cells. In addition, Foxp3+ cells were elevated in lung tissue of Urethane-injected mice [1, 2]. These findings suggest a suppressive status after lung cancer induced by Urethane. On the other hand, there was an increase of toll like receptor 4 (TLR4) expressions in lung tissue and interferongamma (IFN-γ) production ex-vivo by splenocytes suggesting immune response against tumor [1, 2]. We hypothesized that there is an imbalance of effector immune response versus suppression/ tolerance culminating with cancer development.

It has been shown that one of the mechanisms responsible for tumor growth is the increased generation of MDSC in bone marrow followed by their migration to blood and tumor site [3]. These cells act suppressing the immune system through the secretion of factors such as arginase-1 and TGF-β [reviewed in [4, 5]. Another possible role played by tumor cells and/or MDSC is the interference with hematopoiesis [3]. The increase of myelopoiesis can lead to the inhibition in the complete maturation of myeloid cells and thus to the decrease in the number of inflammatory macrophages and elevation in the number of suppressive immature myeloid cells [6]. In agreement, we found an increased percentage of MDSC in bone marrow of mice induced to lung cancer by Urethane administration [2].

Although MDSCs depletion causes less tumor burden and improved immune response in experimental models, this procedure has not been associated with cure [7, 8]. Considering that cancer is a complex and multifactorial disease the treatment also should be based on multiple targets.

A recently described target is the renin angiotesing system (RAS) as some components are proposed to play a role in cancer development [9]. RAS is a network of enzymes and peptides and its key peptidase angiotensin-converting-enzyme (ACE, EC.3.4.15.1) cleaves several substrates including bradykinin (B). The intrarenal hormone bradikynin modulates renal functions [10,11] and renal cells growth and proliferation [12, 13] exerting its multiple functions via B1 and B2 receptors [14, 15]. In addition to the classic functions of ACE and B1/B2 it has been suggested the participation of these components in cancer pathophysiology including the expression of B1 and B2 in clinical specimens of several cancers such as small cell and non-small cell carcinomas of the lung [16, 17, 18].

Shen et al. using experimental models of tumor and chronic inflammation showed that mice over expressing ACE in myeloid cells present reduced number of MDSCs in blood and spleen [19] which confirms previous findings that ACE is associated with some aspects of myelopoiesis regulation [20, 21]. All these findings strongly suggest the participation of RAS members in cancer development which could occur via modulation of MDSC and increased immunosuppression favoring tumor growth.

In humans there are some conflicting data relating ACE and cancer development. Whereas meta-analysis showed no association of ACE gene insertion/deletion polymorphism and lung cancer development (Wang et al. [22]), the use of angiotensin-converting enzyme inhibitors was associated with an increased rate of lung cancer in a nested case-control analysis (Azoulay et al. [23]). Experimental models have shown that ACE inhibitors can decrease tumor growth as observed by Attoub et al. [24] in athymic mice injected with highly tumorigenic LNM35 human lung cells (xenografts) and treated with captopril. Moreover, Araujo et al. [25] showed in renal cell carcinoma model that ACE blockade was associated with smaller tumors and fewer lung metastases compared with the controls.

Thus, RAS members (ACE, B1 and B2 receptors) could be used in addition to MDSCs as targets for cancer diagnostic, prediction of outcome, and therapy. In this line, Nowak et al. [26] showed that ACE activity was significantly lower in lung tissue from patients undergoing tumor resection than in individuals with normal lung tissue.

In addition to ACE expression during cancer development the multiple cross talks between bradykinin receptors (B1 and B2) and renin-angiotensin system affecting cancer development have been studied [27] but no evaluation has been performed on bradykinin receptors and MDSC in cancer.

Therefore, our aim was to investigate at the tumor site whether there is an increase in myeloid-derived suppressor cells and the possible correlation with bradikynin receptors and angiotensinconverting enzyme expressions in mice submitted to Urethaneinduced lung cancer.

Material and Methods

Animals and experimental design

Eight to 10-week-old male BALB/c mice (bred in CEDEME/ UNIFESP) were placed in cages and cared for in accordance with the Principles of Laboratory Animal Care (NIH publication 86-23, revised 1985) and the regulations of the Brazilian Committee on Animal Experimentation. The project was submitted and approved by the Animal Ethics Committee of UNIFESP (protocol 0117/09).

Control: Mice were just followed during 120 days (n=5)

MDSC: Mice were adoptively transferred with myeloid-derived suppressor cells and followed during 120 days in order to evaluate whether the increased percentage of MDSC could lead to tumor development (n=5)

UR: (urethane induced-lung cancer) Mice were injected with Urethane and followed during 120 days (n=5).

In order to induce lung cancer Urethane (Sigma Chemical Company, St Louis, MO) dissolved in 0.9% NaCl was injected in BALB/c mice (two doses i.p. with the interval of 48 h; 1.5 g/kg each dose).

Animals were followed for 120 days when they were anesthetized with Xylazine (Agribrands, Brazil) and Ketamine (Vetbrands, Brazil) diluted in 10 ml of sterile PBS (phosphate buffered solution-OXOID LTD Hampshire England) for the harvesting of spleen, blood and lung.

Spleen and blood were evaluated for the percentages of CD11b+ Gr-1+ cells by flow cytometry. Lung lobes were segmented and used for: flow cytometry to evaluate the percentages of CD11b+ Gr-1+ cells, histology (nodules identification and measurement), PCR (mRNA), and immunohistochemistry.

Myeloid-derived suppressor cells enrichment for adoptive transfer

Our goal was to increase the percentage of myeloid-derived suppressor cells (by adoptive transfer) in naïve BALB/c mice to evaluate whether these cells in higher percentage could induce lung cancer development even in the absence of the carcinogen Urethane.

In order to obtain an enrichment of MDSCs for adoptive transfer we injected BALB/c mice (i.p.) twice with 1.5g/kg of Urethane (Sigma Chemical Company, St Louis, MO) dissolved in 0.9% NaCl and with an interval of 48 hours between administrations. Twenty days later mice were anesthetized and lungs were removed for cell harvesting, and a single cell suspension was obtained. Lung cells were stained with CD11b APC (BD Biosciences Pharmingen) and Gr-1 Pe (Immuno- Tools, Germany). Cells were sorted by flow citometry using FACS Aria (BD Biosciences, San Jose, CA). It was obtained 95% of myeloid derived suppressor cells purity (shown below) (Figure 1).