Assessment of Cytokine mRNA Expression Profiles in Tumor Microenvironment and Peripheral Blood Mononuclear Cells of Patients with High-grade Serous Carcinoma of the Ovary

Abstract

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-4-mediated 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.

Cytokine mRNA and protein expression by cell cultures of epithelial ovarian cancer—Methodological considerations on the choice of analytical method for cytokine analyses

“…We analyzed, in paired samples, the protein expression of IL‐1β, IL‐6, IL‐8, TNF‐α, and TNF‐β/LTA in the serum of 14 women, suffering from HGSC, and compared it to previously analyzed mRNA expression in biopsies from their tumors 6 . The results are summarized in Table 3.…”

“…Studies of cytokines in health and various diseases 1,2 have gained more and more attention, not only due to their significant biological role, but also in the search of biomarkers for diagnosis and treatment 1,3,20 . Despite the fact that the biological effects of cytokines are driven by their protein/peptide form, the assessment of cytokine mRNA expression signatures for defining functional cytokine pathways of pathogenic importance has gained a wide use 6‐12 . Factors influencing the cytokine mRNA and protein correlation are many, both biological and experimental, ranging from gene transcription to protein translation and degradation 21,22 .…”

“…The samples were collected after ethical permission and informed consent. Cytokine mRNA expression in ovarian tumor tissue from the same patients, previously studied and published, 6 was used as comparison to the protein cytokine assessment.…”

“…It has also been an important technique in identifying different diagnostic tests and predicting outcomes for different malignant and non‐malignant diseases 13 . Recently, we used this method to gain knowledge on the cytokine mRNA expression profile in paired ovarian cancer tissue samples and peripheral blood mononuclear cell (PBMC) samples of women suffering from HGSC 6 . Our results indicated elevated cytokine mRNA levels in the ovarian cancer microenvironment compared with non‐malignant conditions and normal ovarian tissue, where inflammation, immune suppression, and promotion of T reg cells prevailed.…”

“…Because of this, a need for alternative methods for revealing cytokine presence has emerged. Real‐time quantitative RT‐PCR is one of the methods often used as a proxy to cytokine protein analyses 6‐12 . It has also been an important technique in identifying different diagnostic tests and predicting outcomes for different malignant and non‐malignant diseases 13 .…”