Glioblastoma cell ability to adapt their functioning to microenvironment changes is a source of the extensive intra-tumor heterogeneity characteristic of this devastating malignant brain tumor. A systemic view of the metabolic pathways underlying glioblastoma cell functioning states is lacking. We analyzed public single cell RNA-sequencing data from glioblastoma surgical resections, which offer the closest available view of tumor cell heterogeneity as encountered at the time of patients’ diagnosis. Unsupervised analyses revealed that information dispersed throughout the cell transcript repertoires encoded the identity of each tumor and masked information related to cell functioning states. Data reduction based on an experimentally-defined signature of transcription factors overcame this hurdle. It allowed cell grouping according to their tumorigenic potential, regardless of their tumor of origin. The approach relevance was validated using independent datasets of glioblastoma cell and tissue transcriptomes, patient-derived cell lines and orthotopic xenografts. Overexpression of genes coding for amino acid and lipid metabolism enzymes involved in anti-oxidative, energetic and cell membrane processes characterized cells with high tumorigenic potential. Modeling of their expression network highlighted the very long chain polyunsaturated fatty acid synthesis pathway at the core of the network. Expression of its most downstream enzymatic component, ELOVL2, was associated with worsened patient survival, and required for cell tumorigenic properties in vivo. Our results demonstrate the power of signature-driven analyses of single cell transcriptomes to obtain an integrated view of metabolic pathways at play within the heterogeneous cell landscape of patient tumors.

Cell motility is critical for tumor malignancy. Metabolism being an obligatory step in shaping cell behavior, we looked for metabolic weaknesses shared by motile cells across the diverse genetic contexts of patients’ glioblastoma. Computational analyses of single-cell transcriptomes from thirty patients’ tumors isolated cells with high motile potential and highlighted their metabolic specificities. These cells were characterized by enhanced mitochondrial load and oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). Functional assays with patients’ tumor-derived cells and -tissue organoids, and genetic and pharmacological manipulations confirmed that the cells depend on enhanced ROS production and MPST activity for their motility. MPST action involved protection of protein cysteine residues from damaging hyperoxidation. Its knockdown translated in reduced tumor burden, and a robust increase in mice survival. Starting from cell-by-cell analyses of the patients’ tumors, our work unravels metabolic dependencies of cell malignancy maintained across heterogeneous genomic landscapes.

Cancer cells in similar functional states are found in all glioblastoma, despite the genomic heterogeneity observed between and within these brain tumors. Metabolism being downstream of all signaling pathways regulating cell behaviors, we looked for metabolic weaknesses in link with motility, a key functional state for glioblastoma aggressiveness. A signature-driven data reduction approach highlighted motile cells present in thirty tumors from four independent single-cell transcriptomic datasets. Analyses integrating trajectory modeling disclosed, as characteristic of motile cells, enhanced oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). The soundness of this prediction was verified using migration and invasion assays with patient-derived cells and tissue organoids. Pharmacological and genetic manipulations showed that enhanced ROS production and MPST activity are required for glioblastoma cell motility. Biochemical assays indicated that MPST acts by protecting protein cysteine residues from dismal hyperoxidation. In vivo, MPST knockdown translated in reduced tumor burden, and a robust increase in mice survival. These results show that enhanced oxidative stress coupled with MPST mobilization plays a key role in glioblastoma cell motility.

Inflammatory cytokines perturb hematopoietic stem cell (HSC) homeostasis and modulate the fitness of neoplastic HSC clones in mouse models. However, the study of cytokines in human hematopoiesis is challenging due to the concerted activities of multiple cytokines across physiologic and pathologic processes. To overcome this limitation, we leveraged serial bone marrow samples from patients with CALR-mutated myeloproliferative neoplasms who were treated with recombinant interferon-alpha (IFNa). We interrogated baseline and IFNa-treated CD34+ stem and progenitor cells using single-cell multi-omics platforms that directly link, within the same cell, the mutation status, whole transcriptomes and immunophenotyping or chromatin accessibility. We identified a novel IFNa-induced inflammatory granulocytic progenitor defined by expression and activities of RFX2/3 and AP-1 transcription factors, with evidence supporting a direct differentiation from HSCs. On the other hand, IFNa also induced a significant B-lymphoid progenitor expansion and proliferation, associated with enhanced activities of PU.1 and its co-regulator TCF3, as well as decreased accessibility of megakaryocytic-erythroid transcription factor GATA1 binding sites in HSCs. In the neoplastic hematopoiesis, the lymphoid expansion was constrained by a preferential myeloid skewing of the mutated cells, linked with increased myeloid proliferation and enhanced CEBPA and GATA1 activities compared to wildtype cells. Further, IFNa caused a downregulation of the TNFa signaling pathway, with downregulation of NFKB and AP-1 transcription factors. Thus, IFNa simultaneously initiated both - pro-inflammatory and anti-inflammatory - cell states within the same hematopoiesis, and its phenotypic impact varied as a function of the underlying HSC state and mutation status.

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