Dysregulation of Peripheral Blood Mononuclear Cells and Immune-Related Proteins during the Early Post-Operative Immune Response in Ovarian Cancer Patients

Abstract

Surgical treatment is a cornerstone of ovarian cancer (OC) therapy and exerts a substantial influence on the immune system. Immune responses also play a pivotal and intricate role in OC progression. The aim of this study was to investigate the dynamics of immune-related protein expression and the activity of peripheral blood mononuclear cells (PBMCs) in OC patients, both before surgery and during the early postoperative phase. The study cohort comprised 23 OC patients and 20 non-cancer controls. A comprehensive analysis of PBMCs revealed significant pre-operative downregulation in the mRNA expression of multiple immune-related proteins, including interleukins, PD-1, PD-L1, and HO-1. This was followed by further dysregulation during the first 5 post-operative days. Although most serum interleukin concentrations showed only minor changes, a distinct increase in IL-6 and HO-1 levels was observed post-operatively. Reduced metabolic and phagocytic activity and increased production of reactive oxygen species (ROS) were observed on day 1 post-surgery. These findings suggest a shift towards immune tolerance during the early post-operative phase of OC, potentially creating a window for treatment. Further research into post-operative PBMC activity could lead to the development of new or improved treatment strategies for OC.

Keywords: HO-1; IL-10; IL-1β; IL-4; IL-6; PBMC; PD-1; PD-L1; ovarian cancer; surgery.

Figure 1

Blood sampling and assay schedule for the ovarian cancer (OC) patients. The lower section of the timeline shows the four specific time points for blood collection. The upper section of the figure outlines the assays conducted at each blood sampling: RT-PCR, real-time polymerase chain reaction; WB, Western blot assay; function, assessment of peripheral blood mononuclear cells (PBMC) metabolic and functional activity; Luminex assay; ELISA assay.

Figure 2

The mRNA expression of interleukins, HO-1, PD-1, and PD-L1 was significantly downregulated in the PBMCs of OC patients before surgery, with the exception of IL-10. The histogram shows the relative expression of mRNA compared to healthy controls, with the values normalised to one. * p > 0.05; ** p = 0.02; *** p ≤ 0.001.

Figure 3

Surgical treatment alters the expression of interleukin mRNAs in the PBMCs of OC patients. The relative expression levels of different interleukin mRNAs are shown at days 1, 3, and 5 post-surgery compared to the pre-surgical baseline. All p-values comparing the different days were >0.05 unless otherwise specified. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 4

Surgical treatment causes dysregulation of HO1, PD-1, and PD-L1 expression in PBMCs from OC patients. (A) Relative expression of HO-1, PD-1, and PD-L1 mRNAs at days 1, 3, and 5 after surgical treatment compared to the pre-surgical baseline. (B) Changes in the HO-1 protein level in PBMCs (left), as determined by densitometric scanning of immunoblots and normalised to GAPDH (representative immunoblot on the right). All p-values comparing samples from different days were >0.05 unless otherwise specified. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 5

PBMC activity in healthy controls and OC patients before and after surgical treatment. Functional activity (right panel) encompassed the evaluation of nitric oxide (NO) and reactive oxygen species (ROS) production, alongside measuring phagocytic activity (PHAG). Metabolic activity (left panel) represents PBMCs viability assessed via the resazurin metabolism assay (AlamarBlue) without an activator, and subsequently repeated with the same activators used in the functional activity assays (LPS, lipopolysaccharide; L-arginine; TBHP, tert-butyl hydroperoxide). The relative activity shown was computed by comparing fluorometric or spectrophotometric assay results between observations with and without activators. Resazurin metabolism without activators was assessed using a fluorometric assay and then normalised to the control group. All p-values between groups are >0.05 unless stated otherwise. * p = 0.02; ** p ≤ 0.01; *** p = 0.001.

Figure 6

Changes in serum concentrations of interleukins, HO-1, PD-1, and PD-L1 in pre- and post-operative OC patients. (A) Serum levels of interleukins evaluated using Luminex assay. (B) Serum levels of PD-1, PD-L1, and HO-1 evaluated by the ELISA method. All p-values between groups are >0.05 unless stated otherwise. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001.