Chemotherapy promotes antigen presentation in monocytes of patients with high-grade serous ovarian carcinoma [RRBS]

The major aim of our study was to uncover tumor-specific transcriptional and epigenetic changes in peripheral blood monocytes in patients with high-grade serous ovarian cancer (HGSOC). Another key point was to elucidate how chemotherapy can reprogram monocytes and how that relates to changes in other immune subpopulations in the blood. To this end, we performed single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) from patients with HGSOC who underwent neoadjuvant chemotherapeutic treatment (NACT) and in treatment-naïve patients. Monocyte cluster was significantly affected by tumor-derived factors as well as by chemotherapeutic impact. Bioinformatical analysis revealed three distinct monocyte subpopulations within PBMCs based on feature gene expression – CD14.Mn.S100A8.9hi, CD14.Mn.MHC2hi and CD16.Mn subsets. The intriguing result was that NACT induced antigen presentation in monocytes by the transcriptional upregulation of MHC class II molecules, but not by epigenetic changes. Increased MHC class II gene expression was a feature observed across all three monocyte subpopulations after chemotherapy. Our data also demonstrated that chemotherapy inhibited interferon-dependent signaling pathways, but activated some TGFb-related genes. Our results can enable personalized decision regarding the necessity to systemically re-educate immune cells to prime ovarian cancer to respond to anti-cancer therapy or to improve personalized prescription of existing immunotherapy in either combination with chemotherapy or a monotherapy regimen. For RRBS profiling, CD14+ circulating monocyte samples were collected from patients with ovarian cancer (n=8) and healthy donors (n=6). Ovarian cancer samples were divided according to treatment plan: 3 patients received platinum-based neoadjuvant chemotherapy (NACT) [carboplatin plus paclitaxel] prior to surgery and 5 patients had debulking surgery without upfront NACT. After surgery all patients received platinum/taxane-based adjuvant chemotherapy. The peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation using Lymphosep, Lymphocyte Separation Media (#L0560-500, Biowest, France), 1.077 g/ml density, at 600g for 30 minutes. The isolation of monocytes from PBMCs was performed using positive magnetic selection with CD14+ MACS beads (#130-050-201, Miltenyi Biotech, Germany), resulting in 90–98% monocyte purity as confirmed by flow cytometry. After monocyte isolation, the samples were washed twice with DPBS without calcium and magnesium at 300 g for 10 minutes. Subsequently, the cell precipitate was lysed using lysis buffer RLT (#79216, Qiagen, USA) and stored at -80°C until further experiments. Lysed monocytes were used for DNA isolation and reduced representation bisulfite sequencing (RRBS-Seq). DNA were extracted from lysed monocyte samples using AllPrep DNA/RNA/miRNA Universal Kit (#80224, Qiagen, USA). The quality of DNA was assessed by TapeStation 4150 automated electrophoresis system (#RRID: SCR_019393, Agilent Technology, USA). The quantity of DNA was assessed by Qubit 4 fluorometer (#RRID: SCR_018095, ThermoFisher Scientific, USA). Samples were purified using magnetic beads AMPure XP (Beckman Coulter) and concentration was controlled using Fluoroskan. Libraries were prepared using Zymo-Seq RRBS Library Kit (#D5461) and Zymo-Seq UDI Primer Plate (#D3096) (Zymo Research, USA), according to the manufacturer’s instructions. Sequencing was performed by the Illumina NovaSeq 6000 platform (Illumina, USA).