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.
Overall design: For scRNAseq profiling, peripheral whole-blood samples were collected from patients with ovarian cancer (n=6) 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 3 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. PBMCs were washed twice with DPBS without calcium and magnesium at 300 g for 10 minutes and counted using CytoFLEX flow cytometer (Beckman Coulter, USA). 1 000 000 PBMCs were used to prepare cell suspension for single-cell sequencing. Cells were put into 1.5 ml cryo tubes and mixed with 500 μl of X-VIVO™ 10 Medium (#180989, Lonza, Switzerland), 400 μl of fetal bovine serum (#10500064, ThermoFisher Scientific, USA), and 100 μl of dimethyl sulfoxide (#F135, Paneco, Russia). Subsequently, the cell suspension was stored in cryo container at -80°C for 48 hours, and then stored in liquid nitrogen until further experiments (but not more than 6 months). Single-cell RNA sequencing was performed on the Chromium X platform, using the 10х Genomics Chromium Next GEM Single Cell 3′ Reagent Kit v3.1. (10х Genomics, USA). Prior to library preparation, cells were counted and quality of samples was assessed. Up to 8000 cells were used for further manipulations. cDNA amplification and library construction were conducted following the manufacturer’s protocol. Sequencing was performed with the Illumina Nextseq 2000 platform (Illumina, USA).
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