Assessment of cytokine mRNA expression profiles in tumor microenvironment and peripheral blood mononuclear cells of patients with high-grade serous carcinoma of the ovary

Objective: Tumor establishment, metastatic spreading and poor survival in ovarian cancer is strongly associated with progressive derangement of the patient’s immune system. Accumulating evidence suggests that immune impairment is influenced by the production and presence of cytokines in the tumor microenvironment. Methods: Cytokine mRNA profiles in tumor tissue and peripheral blood mononuclear cells (PBMC) were analyzed in patients with high grade serous carcinoma (HGSC) of the ovary and compared it to patients with benign ovarian conditions and controls with normal ovaries. Cytokine assessment was done by real-time quantitative RT-PCR and specific primers and probes for 12 cytokines-IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-α, TNF-β/LTA, TGF-β1, and GM-CSF chosen to distinguish between cytotoxic Th1, humoral Th2, regulatory Th3/Tr1 and inflammatory responses. Results: The cytokine mRNA response in the HGSC patients was significantly up regulated compared to patients with benign ovarian conditions and normal ovary controls confirming the immunogenicity of HGSC and implying immune recognition and reaction locally in the tumor microenvironment and systemically in the peripheral blood.There was an up-regulation of inflammatory and inhibitory cytokine mRNA promoting tumor progression, T-regulatory cell priming and T-regulatory cell-mediated immune suppression. In contrast, there was an inability to mount the crucially important IFN gamma response needed for upregulation of the cytotoxic anti-tumor response in the local microenvironment. In addition, systemic IL-4mediated Th2 response prevailed in the peripheral blood deviating the systemic defense towards humoral immunity. Conclusions: Taken together, these results suggest local and systemic cytokine cooperation promoting tumor survival, progression and immune escape. Our study confirms and extends previous investigations and contributes to the evaluation of potential cytokine candidates for diagnostic cytokine mRNA profiles and for future therapeutic interventions based on cytokine inhibition. late stage and are genetically unstable. Since ovarian cancer has diffuse symptoms and there is no specific diagnostic marker(s), most women are diagnosed with metastasized disease (FIGO stages III and IV) [2]. Cytokines, modulating immune responses, may play a significant role in the establishment and progression of ovarian cancer [8]. Cytokines are cell-signaling proteins/peptides for intercellular communication, secreted by a variety of cells. Different cytokine profiles, designated Th1, Th2, Th3, Tr1 and Th17, are associated with the ability to mediate and regulate immunity and inflammation, promote or halt cell growth and movement or immune responses. Thus, a cytokine profile dominated by IFN-γ and interleukin (IL)-15 (Th1) promotes cytotoxicity, a cytokine Citation: Israelsson P, Labani-Motlagh A, Nagaev I, Dehlin E, Nagaeva O, et al. (2017) Assessment of Cytokine mRNA Expression Profiles in Tumor Microenvironment and Peripheral Blood Mononuclear Cells of Patients with High-grade Serous Carcinoma of the Ovary. J Cancer Sci Ther 9: 422-429. doi: 10.4172/1948-5956.1000453 J Cancer Sci Ther, an open access journal ISSN: 1948-5956 Volume 9(5)422-429 (2017) 423 conditions and 8 with normal ovaries. Information about the patients and the histopathologic diagnosis according to the World Health Organization Classification [18], was extracted from their medical records. Preparation of tissue and blood samples Tissue samples, snap-frozen in liquid nitrogen and stored at -150°C, and corresponding Buffy coats from the patients with HGSC, benign ovarian conditions and normal ovaries were retrieved from the Ovarian Cancer Biobank at Norrland’s University Hospital. PBMC were isolated from Buffy coats by Lymphoprep (Nycomed) gradient centrifugation as previously described [19,20]. The collected interphase containing lymphocytes and macrophages was washed, counted, frozen and kept at -80°C until use. Total RNA extraction and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) Cytokine gene expression analysis was performed by real time RT-qPCR following the MIQE requirements [21]. A summary of the positive samples from the total number of samples for the individual cytokines analyses are given in Table 1. RNA extraction: About 0.5 mg of frozen ovarian tissue/sample was sliced into 25 μm slices in a cryostatic microtome, dissolved in 350 μl of lysis buffer and total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). PBMC from Buffy coats were thawed by layering 200 μl RNA later stabilization solution (AM7020, ThermoFisher Scientific inc., USA) mixed by pipetting and transferred to a tube with TRIzol Reagent (ThermoFisher Scientific). Total RNA was isolated and cleaned up with RNeasy Mini kit. RNA yield (on average 3961 ng in total volume of 50 μl) and purity (average A260/A280=1.7) were assessed by spectrophotometry (NanoDrop, ThermoScientific). Reverse transcription: For each sample 400 ng total RNA in a reaction volume of 20 μl was transcribed into cDNA by random hexamers, MuLV reverse transcriptase and dNTP mix using highcapacity cDNA reverse Transcription Kit (ThermoFisher) according to the manufacturer’s description. Thereafter, 60 μl sterile miliQ water was added to each sample to adjust the cDNA concentration to 5 ng/ μl total RNA. profile dominated by IL-4 (Th2) promotes humoral immunity, while IL-17 (Th17), IL-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and TNF-β/LTA promote inflammation, and TGF-β and IL-10 (Th3, Tr1) promote T regulatory cell-induced immunosuppression. These cytokines can be expressed in small amounts in the normal ovaries as well [8]. Previous assessments of protein concentration of cytokines in serum and ascites of EOC patients and in supernatants of EOC cell lines have shown that there is a shift towards immunoinhibitory cytokines [8-13]. Additionally, in earlier reports from the nineties, attempts were made to assess mRNA expression for a limited number of individual cytokines in tumor tissue using Northern blot or semi-quantitative RTPCR [14-17]. Here, we quantified the mRNA transcription of a broad number of cytokines in paired samples of tumor tissue and peripheral blood mononuclear cells (PBMC) from HGSC, benign ovarian conditions and normal controls by real-time RT-qPCR. The cytokine primers and probes used distinguish between the main cytokine mRNA patterns, i.e. Th1-, Th2-, Th3/Tr1-and inflammatory pattern, known to be the key cytokines that control cytotoxic-, humoral-, regulatoryand inflammatory responses respectively. Our goal was to elucidate the local cytokine mRNA production in the tumor tissue and to compare it to the systemic cytokine profile measured in PBMC of the same patients as well as to compare it to benign ovarian conditions and normal ovaries aiming to reveal differences in the systemic and local modulation of the patient’s immune response to HGSC. Materials and Methods