Interleukin-6 Induced Acute Phenotypic Microenvironment Promotes Th1 Anti-Tumor Immunity

Ting Xue1, Ping Liu1, Yong Zhou2, Kun Liu1, Li Yang2, Robert L. Moritz2, Wei Yan1, Lisa X. Xu1

1. Key Laboratory of Systems Biomedicine (MOE), Shanghai Center for Systems Biomedicine, the School of Biomedical Engineering and MED-X Research Institute, Shanghai Jiao Tong University, Shanghai, China.

2. Institute for Systems Biology, Seattle, WA, USA.

 Corresponding author: WY: weiyan@sjtu.edu.cn, Tel: +86-21-34201452; LXX: lisaxu@sjtu.edu.cn, Tel: +86-21-62932302; RLM:

robert.moritz@systemsbiology.org, Tel: (206) 732-1244.

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Received: 2015.11.11; Accepted: 2016.02.08; Published: 2016.03.21

Abstract

Cryo-thermal therapy has been emerged as a promising novel therapeutic strategy for advanced breast cancer, triggering higher incidence of tumor regression and enhanced remission of metastasis than routine treatments. To better understand its anti-tumor mechanism, we utilized a spontaneous metastatic mouse model and quantitative proteomics to compare N-glycoproteome changes in 94 serum samples with and without treatment. We quantified 231 highly confident N-glycosylated proteins using iTRAQ shotgun proteomics. Among them, 53 showed significantly discriminated regulatory patterns over the time course, in which the acute phase response emerged as the most enhanced pathway. The anti-tumor feature of the acute response was further investigated using parallel reaction monitoring target proteomics and flow cytometry on 23 of the 53 significant proteins. We found that cryo-thermal therapy reset the tumor chronic inflammation to an “acute” phenotype, with up-regulation of acute phase proteins including IL-6 as a key regulator. The IL-6 mediated “acute” phenotype transformed IL-4 and Treg-promoting ICOSL expression to Th1-promoting IFN-γ and IL-12 production, augmented complement system activation and CD86+MHCII+ dendritic cells maturation and enhanced the proliferation of Th1 memory cells. In addition, we found an increased production of tumor progression and metastatic inhibitory proteins under such “acute” environment, favoring the anti-metastatic effect. Moreover, cryo-thermal on tumors induced the strongest “acute” response compared to cryo/hyperthermia alone or cryo-thermal on healthy tissues, accompanying by the most pronounced anti-tumor immunological effect. In summary, we demonstrated that cryo-thermal therapy induced, IL-6 mediated “acute” microenvironment shifted the tumor chronic microenvironment from Th2 immunosuppressive and pro-tumorigenic to Th1 immunostimulatory and tumoricidal state. Moreover, the magnitude of “acute” and “danger” signals play a key role in determining the efficacy of anti-tumor activity.

Key words: “acute” phase response, cryo-thermal therapy, interleukin-6, parallel reaction monitoring, Th1anti-tumor immunity.

Introduction

Metastatic breast cancer accounts for the most death for women with an overall 5-year survival rate

of approximate 24% [1]. Although current systemic treating approaches such as chemotherapy and target therapy have significantly improved the survival rate of patients, more improvements in therapeutic strategies are desirable towards alleviating the cancer-related symptoms, improving patients’ life qualities, as well as extending their survival periods. Recently, thermal therapies including thermal ablation (> 60°C) and cryosurgery have emerged as intervention strategies and been applied as alternative or adjuvant clinical treatments on metastatic breast cancer, taking advantages of their minimal invasiveness, local targeting and less side effects [2, 3]. The cryo/thermal ablation therapy, a combinatorial variant of the thermal strategies, was reported to generate maximal destruction and larger ablation zones [4, 5], considerable decrease of tumor recurrence [6] and a complete tumor regression rate of 41%, providing a superior approach over thermal ablation or cryosurgery alone [7]. Our group has been working on a novel cryo/thermal system for years [8, 9] and previously reported its application for metastatic breast cancer treatment in animal models. By combinatorial usage of liquid nitrogen and radio frequency, this system incurs a rapid temperature shift between freezing (-20 °C) and heating (50°C) on local primary tumors and thereby delivers a promising therapeutic efficacy with an increased survival rate (60%-70%), free-recurrence and significantly reduced metastasis (70%) [10-12].

Hyperthermia or cryosurgery alone was previously reported to induce tumor cell death via cell membrane destruction, cytotoxicity and coagulative necrosis [13], activating a concomitant immune response [14, 15]. Nevertheless, the biological mechanism has not been comprehensively studied. In the past,, we indicated that cryo-thermal therapy increased the damage region of tumor, and led to severe destruction of tumor blood vessels [8, 16]. Furthermore, we observed an increased local induction of Th1 type cytokines and recruitment of CD4+ and CD8+ T lymphocytes [10, 17], while the accumulation of immunosuppressive lymphocytes were drastically reduced [10, 18]. However, cryo-thermal therapy appears to induce more systematic and profound responses in vivo, which therefore appeals for a comprehensive and system-wide analysis towards better understanding the anti-tumor mechanism at a molecular level.

Mass spectrometry based proteomics provides such a powerful tool to systematically study protein proprieties and profiles in a complex biological system [19]. To date, high throughput proteomics involves two main strategies. The first is referred as “shotgun” or “discovery” proteomics characterized by data-dependent acquisition (DDA) on mass spectrometer, allowing detection of thousands of proteins per analysis. The other is called “target” proteomics, which permits reproducible quantification of targeted proteins in large scale samples (e.g. clinical samples). Among the target proteomics approaches, selected reaction monitoring (SRM) is the first generation and stands as the golden standard so far, which was successfully applied in clinical biomarker discovery [20]. Recently, a new targeted method, called parallel reaction monitoring (PRM), operated in the high resolution and high mass accuracy spectrometers (e.g. Q-Exactive) provides an alternative to SRM [21, 22]. Different from SRM, in which fragments per peptide (termed as transitions) are monitored once at a time, PRM allows simultaneous monitoring of all transitions as a full MS/MS scanning profile and thus provides an enhanced selectivity and confidence in quantitation of each analyzed target protein [23].