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ScRNA-seq of HGSC tumor and ascites

ABSTRACT: Immune regulatory metabolites are key features of the tumor microenvironment (TME), yet with a few notable exceptions, their identities remain largely unknown. We uncovered the immune regulatory metabolic states and metabolomes of sorted tumor and stromal, CD4+, and CD8+ cells from the tumor and ascites of patients with high-grade serous ovarian cancer (HGSC) using high-dimensional flow cytometry and metabolomics supplemented with single cell RNA sequencing. Flow cytometry revealed that tumor cells show a consistently greater uptake of glucose than T cells, but similar mitochondrial activity. Cells within the ascites and tumor had pervasive metabolite differences, with a striking enrichment in 1-methylnicotinamide (MNA) in T cells infiltrating the tumor compared to ascites. Despite the elevated levels of MNA in T cells, the expression of nicotinamide N-methyltransferase, the gene encoding the enzyme that catalyses the transfer of a methyl group from S-adenosylmethionine to nicotinamide, was restricted to fibroblasts and tumor cells. Treatment of T cells with MNA resulted in an increase in T cell-mediated secretion of the tumor promoting cytokine tumor necrosis factor alpha. Thus, the TME-derived metabolite MNA contributes to an alternative and non-cell autonomous mechanism of immune modulation of T cells in HGSC. Collectively, uncovering the tumor-T cell metabolome may reveal metabolic vulnerabilities that can be exploited using T cell-based immunotherapies to treat human cancer.

PROVIDER: EGAS00001004829 | EGA |

REPOSITORIES: EGA

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Project description:Ovarian cancer (OC) ranks among the deadliest cancers in women. Lack of symptoms, rapid spread of metastases and common chemoresistance contribute to unfortunate fate of majority of OC patients, especially those having high-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of OC. Many HGSC patients have excess fluid in the peritoneum at the stage of diagnosis called ascites. Ascites is basically a tumor microenvironment (TME) in the liquid form containing various cells, proteins and also extracellular vesicles (EVs). EVs are small membrane-bound particles that convey proteins, lipids and nucleic acids between cells and their cargo reflects the cell of origin. EVs play important role in cancerogenesis and hold great promise as disease biomarkers as well as potential therapeutic targets. Small size and polydispersity of EVs bring various challenges to their isolation and characterization, including method-dependent enrichment of different EV subtypes as well as contaminants. We isolated EVs from ascites of 11 patients by two different methods (ultracentrifugation coupled to sucrose cushion and size-exclusion chromatography, qEV column) and analyzed them by mass spectrometry. We identified core ascitic EV proteins present in all patients that contain typical EV markers and are devoid of method-dependent contaminants. To cover interpatient heterogeneity, we expanded this “core proteome” with proteins found in majority of patients. Next, we compared them with proteins of EVs from related control fluids and uncovered proteins present only on EVs from HGSC patients. We believe this list of proteins contain both important players of HGSC progression as well as potential biomarkers. Using single cell RNA sequencing data, we mapped the origin of EVs to different types of cells present in malignant ascites. Our results suggest that EVs in ascites do not come predominantly from tumor cells, but rather from variety of non-malignant cell types including cancer-associated fibroblasts and tumor-associated macrophages, which presence in ascites was confirmed by flow cytometry. This emphasizes the recently appreciated role of TME in the progression of HGSC. To conclude, this is the first study attempting to link EV composition to the cell types producing it. As such it opens numerous avenues both for better understanding of EV role in tumor promotion/prevention and for the improved HGSC diagnostics.

Project description:Tumor progression and metastasis are promoted by ascites which constitutes a central part of the ovarian cancer (OC) tumor microenvironment (TME). One prominent immune cell type in ascites is represented by tumor-associated macrophages (TAMs) which contribute to tumor progression. Among other cells, ascites is highly enriched in soluble factors, as well as extracellular vesicles (EVs). It remains relatively unknown, how far TAMs contribute to the EV compartment of the TME. In this work, peripheral blood monocytes from healthy donors were differentiated into monocyte-derived macrophages (MDMs) and polarized into classically activated (M1-like), alternatively activated (M2-like) and TAM-like (by ascites incubation). For all subtypes, serum-free conditioned medium was collected in for 24h and EVs were isolated and characterized by nano-flow cytometry (nFC), label-free mass spectrometry and electron microscopy, among others. Our results demonstrated distinct traits for EV release and cargo across different macrophage subtypes. Particularly, TAM-like MDMs displayed a handicapped small EV release, supported by a total low particle concentration with low frequency of tetraspanin-positive events. These EV subpopulations displayed sizing profiles closer to M1-like than M2-like particles. Also, the low particle release in these cells was supported by truncated expression of EV biogenesis markers (i.e., FLOT-1, TSG101) and a decreased N-glycosylation of CD63 protein in TAM-like MDMs, which was validated in patient-derived samples. Remarkably, the EV-associated proteome of TAMs, displayed significant enrichment in both pro- and anti-inflammatory molecules with clinical value. Interestingly, markers significantly enriched in the ascites TAM-EV signature were mostly associated with poor prognosis, whereas M1-like EV-related markers (pro-inflammatory) were mostly associated with longer survival. Finally, our results confirmed previous data for CD163 and MRC1 to be associated to TAM-EVs, while also describing novel EV-associated candidates to present both diagnostic (i.e., COLEC12) and prognostic (i.e., MRS1) value at circulating levels. Taken together, our data support a unique secretory profile of TAMs in OC providing new biomarkers with translational impact. As for the particularly low particle release, the biogenesis routes involved and the mechanisms behind TAM-EV function need to be further investigated to gain a better understanding of the participation of these cells in the TME landscape.

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ScRNA-seq of HGSC tumor and ascites

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